High throughput system for producing recombinant viruses using site-specific recombination

ABSTRACT

Disclosed are the methods for producing recombinant viruses using site-specific recombination in vitro. In the present invention, circular viral genomic DNAs are digested with restriction enzymes to generate a linear form viral genomic DNAs flanked by site-specific recombination sites, and then are subjected to site-specific recombination with the desired genomic materials flanked by site-specific recombination sites in vitro. According to the present invention, since the site-specific recombination mixture can be applied to host cells without further procedures of selecting the desired recombinant viral genomic DNAs, it is possible to obtain numerous recombinant viruses rapidly at the same time. Thus, the present invention can be used as a high throughput system for generating and screening hundreds or thousands of recombinant viruses.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a method for producing recombinantviruses and vectors for the same. Furthermore, this invention relates toDNA constructs for the method and the recombinant viruses produced bythe method.

[0003] 2. Description of the Related Art

[0004] A recombinant viral vector means a genetically engineered vector,which is used in, for example: the preparation of a vaccine; analysis offunction of a gene, a group of genes, or a genomic domain; production ofproteins; and gene therapy. Genomic materials, which could be integratedinto a viral vector, include any genomic materials such as a gene, cDNA,genomic DNA, DNA sequences encoding peptides or proteins, RNA,anti-sense RNA, siRNA, DNA for si RNA, promoter, enhancer etc. A virus,which is used in the preparation of a vector, includes baculovirus,adenovirus, adeno-associated virus (AAV), retrovirus, Herpes virus,hepatitis B virus (HBV), polioma virus, sindbid virus, and vacxnia virusetc.

[0005] Retrovirus is known as one of the most widely used viruses ingene therapy [Clive Patience et al. J Virol, 72:2671-2676, 1998,Marshall, Science, 269: 1050-1055,1995]. Retroviral genome has two LTRs,capsid sequences and 3 (three) coding regions (gag, pol and env), andthe method of its preparation and its use in vitro and in vivo aredisclosed (see WO9908692A1 and EP 453242). The clinical application ofretroviral vector, however, has the following disadvantages: it forms areplication-competent retrovirus (RCR) (Sharon K. Powell et al. J.Virol., 73:8813-8816, 1999); it has relatively lower level of geneexpression, and the level decreases continuously in vivo; and the titerof virus is low. Furthermore, the retrovirus cannot transfer a gene to anon-dividing cell [Jaffe, E. M. Cancer Res., 53:2221-2226, 1993; Bender,M. A. et al. J. Virol., 61:1639-1646, 1987].

[0006] Adenovirus has a linear genome (sized about 36 kb) and includesreplication origin and capsid signals near to the left inverse terminalregion (ITR) (103 bp) [Shenk, Adenoviridae: The Viruses and TheirReplication. In: Fields B N, Knipe D M Howley P M, ed. Virology.Philadelphia: Raven publishers, 1996: 2111-2148]. Usually, adenoviralvectors have been made by deleting E1 site of the viral genome, which isessential for the viral replication. The viruses including foreigngenomic materials substituting E1 site, are transferred to the packagingcells that provide E1 proteins. Thereby, the viral replication occursonly in the packaging cells. Representative packaging cells include, forexample, HEK293 cell, 911 cell, and PER. C6 cell, etc. [Hitt M M et al.,Advances in Pharmacology, vol. 40, 137-206 (1997), Wang Y. and Huang S.,Drug]. Adenoviral vector has advantages such as safty, affinity tovarious cells, possibility of infecting dividing cells and higher titer(10¹¹ pfu/ml). Thus, adenovirus has been used in the preparation of avector expressing heterologous genes.

[0007] Adeno-associated virus (sized about 4700 bp) has ITRs (sizedabout 145 bp) as replication origins at each terminus. It can beintegrated into the genomic DNAs of various types of host cells safelyand specifically.

[0008] In order to use a recombinant virus as a vector, it should bemodified not to replicate themselves in the infected cells. Therefore,defective viruses, which have deletions of some essential regions ofgenome for viral replication, are usually employed in the preparation ofthe recombinant viral vectors. As for a retrovirus, for example, gag,pol and/or env genes are deleted, and the regions are replaced withdesired genomic materials [Bender et al., J. Virol. 61 (1987) 1639].Regarding an adenovirus, E1, E2 and/or E4 sites are deleted for thepreparation of a viral vector [Levrero et al., Gene 101 (1991) 195;Gosh-Choudhury et al., Gene 50 (1986) 161, Van der Vliet et al.,(1975)]. On the other hand, a pseudo-virus vector, which consists ofonly the essential regions (such as, ITR and capsid sequences) of thereplication, has been used also for obtaining recombinant viruses (seeWO95/02697).

[0009] In addition, baculoviruses, which are known as having circulargenomic DNAs, have been used for the preparation of viral vectors.Baculoviridae are infectious on invertebrates, such as insects andcrustacea. AcNPV is one of the most widely used baculoviruses, and itcontains double strand circular genomic DNAs (sized about 134 kb)(GenBank: NC_(—)001623). Smith et al. developed recombinantbaculoviruses by inserting β-interferon genes, and successfully obtainedinterferon proteins from insect cells using the recombinantbaculoviruses (Smith G E., Mol. Cell. Biol., V3, p2156-2165, 1983).Since then, numerous genes were expressed and produced using recombinantbaculoviral system. The features of the methods are that: i) it ispossible to produce desired recombinant baculoviruses at high efficiencyby employing polyhedrin promoter; and ii) it can be used for theexpression of genes that do not exhibit their activities when incubatedin bacteria. That is because post-translational modification is carriedout in insect cells. However, the size of genome of baculovirus made itdifficult to engineer the genome through conventional restriction andligation method. Therefore, homologous recombination has been used ininsect cells generally. In order for the homologous recombination,carrier vectors containing desired genes and nucleic acid sequencesnecessary for the homologous recombination are prepared. Then, insectcells are transfected with the carrier vectors and viral genomic DNAs toinduce recombination. In that method, however, since the productionefficiency of recombinant viruses is relatively low (0.1-2%), repetitivescreening of baculoviral plaques should be performed in order to obtainthe desired recombinant viruses. Thus, it is also considered as being aninefficient method.

[0010] Kitts P A et al. disclosed an improved method that increasedproduction efficiency of recombinant viruses (Nucleic Acids Res.,October 1990; 18: 5667-5672. Kitts P A et al). They made baculoviralgenomic DNAs to be in linear form using restriction enzyme. In Kitts'method, a linker, which includes Bsu36I recognition base sequences,CCTNAGG, or lacZ genes, is inserted to wild-type Autographa californicanuclear polyhedrosis virus (AcNPV) genome at polyherin locus in order tointroduce Bsu36I site recognition nucleic acid sequences. Next, theviral DNAs are digested with Bsu36I restriction enzyme, and thentransferred to insect cells together with carrier vectors containingdesired genomic materials so as to induce homologous recombination. Theproduction efficiency of the desired recombinant viruses is from 26% to44%. According to that method, linear digested DNA segments, which arenot subjected to recombination, are excluded selectively from theproduction of viral vectors. Thus, the desired recombinant virusesexpressing desired protein could be generated with relatively highefficiency. However, the method has disadvantages as follows: i) it isnot possible to digest Bsu36I recognition sites up to 100% and ii) sincethe digested DNA segments are fused with each other in insect cells byligase, selection procedures for the desired viral vectors need beemployed.

[0011] Peakman T C et al. provided another method using site-specificrecombination of cre/loxP system in vitro (Peakman T C et al., NucleicAcids Research, Vol 20, Issue 3 495-500). Briefly, they preparedbaculoviruses containing loxP sites and applied them to site-specificrecombination in vitro with desired genomic materials by reacting theviruses with transfer vectors having the genomic materials flanked withloxP sites, in the presence of cre recombination enzyme. In that method,time-period for producing recombinant viruses is shortened. However, theproduction efficiency of desired recombinant viruses is still low (0.2%to 49%), thereby it is not suitable to be used as a high-throughputsystem for the preparation of recombinant viruses.

[0012] In addition, Luckow V A et al. reported a method of insertingdesired genomic cassette to baculoviral genome by site-specificrecombination in a cell (Luckow V A et al., Virol., August 1993; 67:4566-4579). According to Luckow's method, firstly, a shuttle vector(sized about 130 kb) is prepared by inserting plasmid replicationorigin, which replicates in bacteria, kanamycin resistance gene andattTn7 recombination sequences into baculoviral genome. The shuttle wasnamed “Bacmid”. DH10Bac was generated by transforming E. coli withBacmid and helper plasmid, which in turn was transformed using a plasmidcontaining desired genes and polyhedrin promoter to prepare recombinantBacmid in E coli. The resulting recombinant Bacmid is isolated andtransferred to insect cells to obtain recombinant viruses. According tothat method, baculoviruses could be obtained with relatively highefficiency by using site-specific recombination of transposon. Thatmethod, however, required complex procedures, for example, cloningdesired genomic materials to Tn7 Bacmid carrier vectors; transformingDH10Bac for the recombination of Tn7 baculoviruses; selecting andisolating bacteria having Bacmid that contains desired genomicmaterials; purifying Bacmid DNAs (sized about 130 kb) from the isolatedbacteria; and transferring the DNAs again to insect cells to producerecombinant virues.

[0013] Thus, non of the above-mentioned methods of the prior arts haveprovided suitable ways to overcome the problems of relatively longtime-period in preparing recombinant viruses (about 3 to 6 weeks) andlow production efficiency. Therefore, there have been demands for thedevelopment of an improved method

[0014] In this regard, we have carried out studies to producerecombinant viruses having desired genomic materials more rapidly andefficiently, thereby we have completed this invention by preparingrecombinant viral vectors in vitro using site-specific recombination andemploying the vectors to produce recombinant viruses. Since the viralvectors obtained in vitro according to this invention can be applied toanimal cells directly without further procedures like selection, thepresent invention not only simplifies the total procedures significantlybut also provides desired recombinant viruses with up to 100% productionefficiency. Thus, higher virus titer can be obtained according to thepresent invention.

SUMMARY OF THE INVENTION

[0015] Any publications referenced herein are hereby incorporated byreference in this application in order to more fully describe the stateof the art to which the present invention pertains.

[0016] It is important to understand the present invention to note thatall technical and scientific terms used herein, unless otherwisedefined, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. The techniques used herein are alsothose that are known to one of ordinary skill in the art, unless statedotherwise.

[0017] Reference to particular buffers, media, reagents, cells, cultureconditions and the like, or to some subclass of same, is not intended tobe limiting, but should be read to include all such related materialsthat one of ordinary skill in the art would recognize as being ofinterest or value in the particular context in which that discussion ispresented. For example, it is often possible to substitute one buffersystem or culture medium for another, such that a different but knownway is used to achieve the same goals as those to which the use ofsuggested method, material or composition is directed.

[0018] It is an object of this invention to provide more rapid andefficient method for producing recombinant viruses.

[0019] In order to accomplish this object, this invention employsrecombinant viral vectors that are prepared in vitro by site-specificrecombination.

[0020] In the present invention, viral genomic DNAs are made in linearform by restriction enzymes before being applied to site-specificrecombination in vitro. Since the linear viral genomic DNAs, which arenot subjected to the site-specific recombination, cannot replicate bythemselves, almost all of the expressed viruses are believed to be thosefrom the circular viral genomic DNAs. Thus, according to the presentinvention, it is possible to obtain recombinant viruses at highefficiency without the procedures for selecting and isolating thedesired recombinant viruses. Accordingly, the method of the presentinvention can be applied to a virus having circular genomic DNAs, forexample, polioma virus, papiloma virus and hepatitis B virus, etc.

[0021] Referring to site-specific recombination, it is an eventoccurring naturally in various living organisms. Enzymes for thesite-specific recombination contain not only endonuclease activity butalso ligase activity, so they recognize a certain part of DNA sequencesand replace it with any other corresponding DNA sequences [Yang W. andMizuuchi K., Structure, 1997, Vol. 5, 1401-1406(9)]. Int/att system frombacterio λ phage, Cre/LoxP system from P1 bacterio phage and FLP-FRTsystem from yeast are well developed site-specific recombinationsystems.

[0022] Site-specific recombination system has been used in i) expressioncontrol of desired genomic materials cloned into recombinant viruses,ii) excising helper-virus when preparing helper-viral dependent viruses,iii) increasing the efficiency of homologous recombination in packagingcells, or iv) inducing site-specific recombination between carriervectors and viral genome in packaging cells to prepare recombinantviruses [Philip N G et al., Biotechniques, vol. 29. 524-528 (2000),Philip N G et al., Human Gene therapy, vol. 10., 2667-2672 (1999), HardyS et al., Journal of Viology, vol. 71., 1842-1849 (1997), Parks R J etal., Proc. Natl. Acad. Sci USA, vol 93., 13565-13570 (1996)]. However,the said applications of the site-specific recombination disclosed inthe prior arts were merely indirect and supplementary uses in vivo or ina cell line, not in vitro.

[0023] In addition, applications of site-specific recombination in thepreparation of recombinant viruses having circular genomic DNAs werereported: i) in the preparation of recombinant baculoviruses in bacteriausing Tn7 recombinantion, and ii) in the preparation of recombinantbaculoviruses using cre/lox-P system in vitro.

[0024] However, there have been no applications of site-specificrecombination in vitro for specific insertion of desired genomicmaterials into viral genomic DNAs in order to obtain recombinant viralvectors. In particular, there have been no applications to prevent theexpression of viral genomic DNAs that do not contain desired genomicmaterials. Thus, the present invention is characterized by employingsite-specific recombination in preparing recombinant viral vectors invitro. The present invention has unexpected advantages compared to theprior arts as follows: i) it is possible to obtain recombinant viralvectors more easily and more rapidly by employing site-specificrecombinant in vitro; ii) it does not require any further procedure ofselecting desired recombinant viruses from intermediate host cells,since the viral genomic DNAs are made in linear form and thereby thelinear viral DNAs, which do not form in circular form by integration ofthe desired genomic materials, cannot be expressed; and iii) it ispossible to generate the same kind of genomic materials from varioustypes of viruses or to generate various genomic materials from the samekind of viral type muti-well (e.g. 96-well or 384-well) by employingcommon transfer vector containing expression cassette that does not haveDNAs from viral genome.

[0025] Furthermore, according to the present invention, homologousrecombination in the packaging cells is not required any more. Nor,particular cell lines for inducing homologous recombination arerequired. Table 1 and Table 2 show the results of the comparisonsbetween the method of the present invention (BacHTS system) and themethods of the prior arts (BacPAK6 system and BacToBac system) withregard to the processes. With the results, it is clear that the desiredrecombinant viruses can be obtained even more rapidly by using themethod of this invention in comparison to the prior arts. TABLE 1 Thecomparisons of the number of procedures between the method of thepresent invention and the methods of prior arts the number of proceduresDays time-period BacPAK6 system 11 6 10 BacToBac system 13 8 12 BacHTSsystem 5 3 7

[0026] TABLE 2 The comparisons of the processes required for preparationof baculoviruses BacPAK system BacToBac system BacHTS system  1. RE cut 1. LR reaction 1. Recombination  2. Agarose gel elution  2.Transformation 2. Transfection  3. Ligaion  3. Colony seeding 3.Infection  4. Transformation  4. Plasmid purification 4. Harvest andvirus storage  5. Colony seeding  5. RE analysis 5. Protein expressionanalysis  6. Plasmid purification  6. DH10Bac ransformation  7. REanalysis  7. Streaking  8. Transfection  8. Seeding  9. Infection  9.Bacmid purification 10. Harvest and virus 10. Transfection    storage11. Infection 11. Protein expression 12. Harvest and virus storage   analysis 13. Protein expression    analysis

[0027] Furthermore, according to the present invention, since promoterfor the expression of the desired genomic materials, and fusion systemand polyadenyl sequences for the preparation of fusion protein areprovided from parent viral genome, the gene cassette cloned to commoncarrier vector could be applied to produce various types of viruses.

[0028] Thus, the present invention provides significantly useful toolsfor studying functions of genes of which demands have been increasinggreatly since the completion of genome project [Wang Y. and Huang S.,Drug Discovery Today, vol. 5, 10-16 (2000)].

[0029] In one embodiment, the present invention employs bacuovirushaving circular genomic DNAs, and Int/att system from bacteriophage λ(Genebank: NC 001416) as a site-specific recombination system. In theInt/att system, attR site-specific recombination sites within viralgenomic DNA fragment react with corresponding attL site-specificrecombination sites including desired genomic materials from carriervector, in the presence of λ integrase recombination enzyme, IHF as aco-factor and Xis, and result in vector DNA fragment containing attB andattP sites [see Hartley J L. Genome Research, 2000, Vol. 10, 1788-1795].Other recombination systems from various organisms, for example, theCre/loxP system from bacteriophage P1, and the FLP/FRT system from theSaccharomyces cerevisiae 2μ circle plasmid, can be employed.

[0030] When the mixture is transferred directly to suitable host cells,which are expected to express desired recombinant viruses, only thecircular viral genomic DNAs containing attB1-[desired genomicmaterials]-attB2 can replicate and form viral particles. The linearviral DNA fragment and the vector DNAs themselves, which do not reactwith each other, and the circular viral DNAs having attP sites cannotreplicate and cannot form viral particles. In FIG. 1, site-specificrecombination between gene cassette and vBacHTS baculoviral DNAs invitro is presented.

[0031] In another embodiment, vBacHTS viral DNAs are constructed toinclude site-specific recombination sequences and Bsu36I recognitionbase sequences, which in turn are made to linear form viral DNAs flankedby 2 (two) site-specific sites with treatment of Bsu36I restrictionenzyme. The linear vBacHTS DNAs cannot form viral particles since thevBacHTS viral DNAs are digested at 2 (two) Bsu36I recognition siteshaving nucleic acid sequences different from each other, while thecircular form DNAs generated by site-specific recombination formed viralparticles efficiently.

[0032] In another embodiment, the reaction mixture containing viral DNAshaving desired genomic materials, which are integrated by site-specificrecombination, is transferred to Sf21 insect cells, and then viralpackaging is detected. Thus, according to the present invention,recombinant viruses can be obtained without employing intermediate hostcells, such as E. coli or yeast, for selecting and amplifying suitablegenomic DNAs. Therefore, the recombinant viruses are suitable for thepurpose of expressing desired genes from host cells. The activitymeasurement of GFP and GUS proteins indicates that the method of thepresent invention is more efficient than the method of the prior arts.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 illustrates gene-screening processes using BacHTS system.

[0034]FIG. 2 depicts the cleavage map of pBacHTS.

[0035]FIG. 3 depicts the cleavage map of pBacHTS2.

[0036]FIG. 4 depicts the cleavage map of pBacPAK8_attR1.

[0037]FIG. 5 depicts the cleavage map of pBacPAK_attR1R2.

[0038]FIG. 6 depicts the cleavage map of pBacHTS_GFP.

[0039]FIG. 7 depicts the cleavage map of pBacHTS_Flag.

[0040]FIG. 8 depicts the cleavage map of pBacHTS_His.

[0041]FIG. 9 depicts the cleavage map of pBacHTS_HisGst.

[0042]FIG. 10 depicts the cleavage map of pBacHTS_Gst.

[0043]FIG. 11 depicts the cleavage map of pBacHTS2_EGFP.

[0044]FIG. 12 depicts the cleavage map of pEntr_EGFP.

[0045]FIG. 13 illustrates the process of preparation of vBacHTS virus.

[0046]FIG. 14 illustrates the process of preparation of bBacHTS2 clone.

[0047]FIG. 15 shows the map of baculoviral polyhedrin locus.

[0048]FIG. 16 illustrates agarose gel electrophoretic analysis ofvBacHTS viral DNAs and bBacHTS2 viral DNAs.

[0049]FIG. 17 illustrates procedures for the preparation of desiredrecombinant viruses using vBacHTS and gene cassette.

[0050]FIG. 18 shows the results of activity assays of GFP baculovirusesand GUS recombinant viruses in vBacHTS system.

[0051]FIG. 19 shows plaque assays for GFP baculoviruses and GUSrecombinant viruses in vBacHTS system.

[0052]FIG. 20 shows the results of activity assays of GFP baculovirusesin bBacHTS2 system.

[0053]FIG. 21 shows the results of activity assays of GUS recombinantbaculoviruses in bBacHTS2 system.

[0054]FIG. 22 illustrates GFP image in multi-well plate and the resultsof western blot.

[0055]FIG. 23 shows enzyme activity using X-Gluc of GUS recombinantviruses in multi-well plate.

[0056]FIG. 24 shows the results of site-specific recombination in vitro.

[0057]FIG. 25 depicts expression of GFP-LacZ fusion proten in Sf21 cellsafter 4 days from the transfer of recombinant DNAs to the cells.

[0058]FIG. 26 shows the results of western blot of the expression ofGFP-LacZ fusion protein using SDS-PAGE and GFP antibodies.

[0059]FIG. 27 depicts LacZ enzyme activity in recombinant virus(GFP-LacZ) using X-Gal substrates.

[0060]FIG. 28 shows the image of Sf21 cells infected with GFP-YPKbaculoviruses from multi-well plate.

[0061]FIG. 29 shows the results of screening of protein kinase (Rb,Histone H1, MBP).

[0062]FIG. 30 depicts phosphorylation of Rb (retinoblastoma) protein.

[0063]FIG. 31 illustrates phosphorylation of Rb protein by #2-A9 proteinkinase.

[0064] Preferred embodiments of this invention are described in thefollowing examples. Other embodiments within the scope of the claimsherein will be apparent to those skilled in the art from considerationof the specification or practice of the invention as disclosed herein.It is intended that the specification, together with the examples, beconsidered exemplary only, with the scope and the spirit of theinvention being indicated by the claims that follow the example. Theexamples herein are meant to exemplify the various aspects of carryingout the invention and not intended to limit the scope of the inventionin any way. The examples do not include detailed descriptions ofconventional methods employed, such as in the performance of genomic DNAisolation, PCR, and sequencing procedures. Such methods are well knownto those skilled in the art and are described in numerous publications.In addition, all the publications referred herein are integrated heretoas references.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Preferred embodiments of this invention are described in thefollowing examples. Other embodiments within the scope of the claimsherein will be apparent to those skilled in the art from considerationof the specification or practice of the invention as disclosed herein.It is intended that the specification, together with the examples, beconsidered exemplary only, with the scope and the spirit of theinvention being indicated by the claims which follow the examples. Theexamples herein are meant to exemplify the various aspects of carryingout the invention and not intended to limit the scope of the inventionin any way. The examples do not include detailed descriptions ofconventional methods employed, such as in the performance of genomic DNAisolation, PCR, and sequencing procedures. Such methods are well-knownto those skilled in the art and are described in numerous publications.In addition, all the publications referred herein are integrated heretoas references.

EXAMPLES Example 1 Preparation of pBacHTS

[0066] pBacHTS viral vectors were prepared. For the preparation,firstly, pBacPAK8_R1 (see FIG. 4) was provided by cloning amplifiedattR1 sites and Bsu36I recognition base sequences at BamHI/EcoRI sitesof a shuttle vector of pBacPAK8 (Genebank: U2446, from Clontech) andtransforming DH5α (from LTI, Life Technologies Inc.) with the clonedvector, and then by incubating and screening in agar medium includingampicilin. Likewise, pBacPAK8_R1R2 was prepared by cloning PCR amplifiedattR2 sites and Bsu36I recognition site sequences at EcoRI/PacI sitesequences of pBacPAK8_R1 (see FIG. 5). In order to obtain pBacHTS, DNAfragment (427 bp) including ccdB gene from EcoR1 digested fragments ofpEntr4 plasmid (from Invitrogen) was inserted to the pBacPAK8_R1R2, andthen the resulting plasmid was transferred to DB3.1 cell (from LTI).pBacHTS contained two different Bsu36I recognition sequences betweenattR1 site and attR2 site (FIG. 2). The attR1 sites and the attR2 sitesincluded site-specific recombinase target sequences that reacted withattL1 and attL2 sites, respectively, in the presence of integrase from λbacteriophage, Xis, IHF-α and IHF-β.

Example 2 Preparation of pBacHTS 6His, pBacHTS GST, pBacHTS HisGst,pBacHTS GFP and pBacHTS Flag

[0067] Fusion tag was inserted to the sites in back of polyhedrinpromoter in order to prepare recombinant baculovirus expressing fusionproteins. pBacHTS_Gst was designed for the preparation of expressionvector for GST fusion protein. In order for this, pGEX-2T(Genbank:U13850) was PCR amplified using a primer including Bg1II and aprimer including BamH1 linker, which in turn was treated with Bg1II andBamHI and then was inserted to pBacHTS/BamHI restriction sites. Inaddition, pBacHTS_His was designed for the preparation of a vectorexpressing His tag fusion protein by inserting 6 His linker to BamHIsite of pBacHTS. Furthermore, pBacHTS_HisGST was designed by insertingBg1II and BamHI treated GST genes, which were PCR amplified, to BamHIsite of pBacHTS_His. On the other hand, GFP gene was PCR amplified usinga primer containing BamHI linker and a primer containing KpnI linker,which in turn was digested with BamHI/KpnI. After that, the digestedgene was integrated into BamHI/KpnI restriction sites of pBacHTS togenerate pBacHTS_GFP. Furthermore, pBacHTS_Flag was prepared byinserting a linker expressing Flag tag at BamHI/KpnI sites of pBacHTS(see FIG. 7).

Example 3 Preparation of DNAs of vBacHTS, vBacHTS His, vBacHTS HisGstand vBacHTS GFP Viruses

[0068] Baculoviruses of which polyhedrin loci were replaced by pBacHTSplasmids were prepared using homologous recombination. Briefly, Sf21insect cells were transfected with a mixture of pBacHTS plasmid, Bsu36Idigested BacPAK6 viral DNAs (from Clontech#6144-1) and Lipofectin (fromLTI) to induce homologous recombination in the cells. The recombinantviruses were isolated through twice repeated plaque assays, which inturn were subjected to PCR. The purified viral clone was named vBacHTSvirus (SEQ. ID No.1: the sequences of its polyhedrin locus) (see FIG.15). As disclosed in FIG. 13, pBacHTS His (see FIG. 8), pBacHTS_HisGst(see FIG. 9), pBacHTS_GST and pBacHTS_GFP were respectively transferredtogether with BacPAK6 virus to generate vBacHTS_His (SEQ. ID No.2: thesequences of its polyhedrin locus), vBacHTS_HisGst (SEQ. ID No.3: thesequences of its polyhedrin locus), vBacHTS_GST (SEQ. ID No.4: thesequences of its polyhedrin locus) and vBacHTS_GFP (SEQ. ID No.5: thesequences of its polyhedrin locus) (see FIG. 15). Viral plaques wereselected from agarose medium.

Example 4 Purification of Viral DNAs of vBacHTS, vBacHTS His, vBacHTSHisGst, vBacHTS GFP, and vBacHTS

[0069] Viral culture medium having 1.2×10⁸ pfu/ml of viral titer wasproduced after 3 days from infecting Sf21 cells on cell culture dish(100 mm diameter) in order to isolate vBacHTS viral DNAs. Then, 1.25×10⁷of Sf21 cells were plated again on cell culture dish (150 mm diameter),which in turn were infected with 20 times larger numbers of viruses(MOI=20). After incubating for 48 hrs, about 25 ml of cell culturemedium per 1 dish was obtained. A total of 150 ml of cell culture mediumfrom 6 dishes (150 mm diameter) was centrifugated at 20,000 rpm for 90min, and the resulting precipitated viral particles were recovered (withHanil, Supra22k). The recovered viral particles were suspended in 2 mlof TE (10 mM Tris-HCl pH=8.0, 1 mM EDTA), and then were treated withsucrose concentration gradient ultra-centrifuge (Beckman SW41 rotor) at30,000 rpm. Viral particles were purified from the layer between 50% and40% sucrose solution. The purified viruses were subjected tocentrifugation again at 18,000 rpm for 90 min. After that, theprecipitates of viral particles were suspended again with 2 ml of TE (10mM Tris-HCl pH=8.0, 1 mM EDTA), and were left at 42° C. for 2 hrs afteradding 0.5% SDS, 1% beta-mercaptoethanol and 0.2 mg of ProteinaseKthereto, so as to isolate viral envelopes. The viral lysate wasextracted twice with the same amount of phenol/chloroform solution, andethanol was added to the extract, thereby pure viral DNAs were obtainedas precipitates. Then, 50 units of restriction enzyme Bsu361 (from NewEngland Bio labs Inc. NEB#524) were added to the purified DNAs (10 ug).The mixture was left at 37° C. for 2 hrs before used in the followingstudies (see FIG. 16).

Example 5 Preparation of Gene Cassette

[0070] A pEntr_GFP gene cassette was prepared by inserting PCR productsof GFP between attL1 site and attL2 site. pEntr_Gus gene cassette fromLTI was employed.

Example 6 Preparation of the Desired Recombinant Viruses Having GFP Geneor GUS Gene

[0071] Recombinant viruses expressing GFP and Gus were generated in thisstudy. The pEntr_GFP gene cassette (100 ng) or the pEntr_Gus genecassette (100 ng) was transferred to a test-tube, and reacted with 100ng of Bsu36I treated vBacHTS viral DNAs at 25° C. for 2 hrs, in thepresence of integrase mixture comprising 4 ul of integrase, Xis, IHF-αand IHF-β. Then, Sf21 cells were infected with the reaction mixturetogether with lipofectin (from LTI), thereby recombinant viruses weregenerated. After 2 days, green fluorescence was observed from the insectcells including GFP gene cassette. Furthermore, after 4 days, almostevery cell showed green fluorescence. Furthermore, fluorescence andsymptoms of viral infection were observed in the infected cellsinfected.

[0072] On the other hand, a mixture containing pEntr_Gus gene cassettewas transferred to Sf21 cells. After 4 days, X-Gluc was added to thecells. After 2 hrs of the reaction, the Sf21 cells became blue, whichsupported the expression of GUS (see FIG. 18). In order to determine theproductivity of the recombinant viruses, 4-day viral culture was dilatedand transferred to insect cells. Next, plaques were detected 4 daysafter adding culture medium containing 1% low-melting point agarose tothe infected cells. As the result, most plaques represented bluefluorescence (see FIG. 19).

Example 7 Determination of the Production Efficiency of RecombinantViruses

[0073] Recombinant viruses expressing GFP were prepared in this study.In order for this, 100 ng of pEntr_GFP gene cassette containing GFPgenes and 200 ng of viral DNAs of Bsu36I treated vBacHTS, vBacHTS_His orvBacHTS_HisGST were reacted in the presence of 4 ul of integrase mixturecomprising integrase, Xis (exisionase), IHF-α and IHF-β at 25° C. for 6hrs. The resulting mixture was transferred to Sf21 cells together withlipofectin (from LTI) in order to generate recombinant baculoviruses.After incubating at 27° C. for 48 hrs, the expression of GFP wasdetected under fluorescence microscope. Since the cells, in whichbaculoviruses replicated, represented fluorescence, the efficiency ofrecombinant viral expression was determined by counting the cellsshowing green fluorescence. In the meantime, BacPAK6 viral DNAs (fromClontech) treated with Bsu36I and 500 ng of pBacPAK_GFP were transferredto Sf21 cells, then the expression of GFP in the Sf21 cells wasmonitored. TABLE 3 The expression efficiency of GFP baculoviruses VirusDNA Bsu36I gene cassette GFP positive cell# BacPAK6 Yes pBacPAK8 EGFP154 VbacHTS pEntr EGFP 1752 VbacHTS yes pEntr EGFP 572 VbacHTS His pEntrEGFP 2236 VbacHTS His yes pEntr EGFP 654 VbacHTS HisGST pEntr EGFP 1433VbacHTS HisGST yes pEntr EGFP 475

[0074] As the result, the efficiency of the viral expression is muchhigher in the method of the present invention compared to the prior arts(see Table 3).

Example 8 Detection of the Efficiency of Recombinant Viral Expression

[0075] Recombinant viruses expressing Gus were prepared, and the numberof plaques expressing Gus genes was counted. In order for this, firstly,100 ng of pEntr_Gus gene cassette containing Gus genes and 200 ng ofeach of viral DNAs of Bsu36I treated vBacHTS, vBacHTS_His orvBacHTS_HisGST were reacted in the presence of 4 ul of integrase mixturecomprising integrase, exisionase, IHF-α and IHF-β at 25° C. for 6 hrs.The resulting mixture was transferred to Sf21 cells together withlipofectin (from LTI) to generate recombinant baculobiruses. Plaqueassays were carried out for the viral culture medium. On the other hand,in order to detect productivity of the recombinant viruses, 4-day viralculture medium was dilated and transferred to insect cells.Subsequently, the cells were incubated for 4 days after adding mediumcontaining 1% low-melting point agarose thereto. The numbers of plaquesexpressing Gus genes were counted after 4 days from the addition of 100ul of 0.33% nutral red water solution and 25 ul of X-Gluc (20 mg/ml inDMSO) and afer subsequent dye treatment (see Table 4). TABLE 4 Theefficiency of GUS recombinant viral expression Re- Virus DNA Bsu36I genecassette Total combinant rate % BacPAK6 yes pBacPAK8 46 41 89% GusVbacHTS pEntr Gus 28 25 89% VbacHTS yes pEntr Gus 57 57 100%  VbacHTSHis pEntr Gus 44 36 82% VbacHTS His yes pEntr Gus 46 45 98% VbacHTSHisGST pEntr Gus 82 57 70% VbacHTS HisGST yes pEntr Gus 32 32 100% VbacHTS GFP pEntr Gus 27 22 81% VbacHTS GFP yes pEntr Gus 21 20 95%

[0076] As the result, most of the plaques represented blue color whenthey were treated with X-Gluc, which supported that the efficiency ofGUS viral generation was very high in comparison to the prior arts.

Example 9 High-Throughput Preparation of Recombinant Viruses inMulti-Well Plate

[0077] Desired recombinant viruses were prepared simultaneously frommulti-well plate. For this purpose, the vBacHTS viral DNAs were treatedwith Bsu36I restriction enzyme. In this study, GFP gene cassette wasemployed to confirm the generation of recombinant baculoviruses and geneexpression. The GFP gene cassettes were incubated in 96-deep well platesimultaneously and purified using automatic device. All the processeswere carried out in multi-well plate while recombination reaction,insect cell incubation, and viral infection were performed using8-channel pipette. About 50 ng of gene cassette and 200 ng of vBacHTSbaculoviral DNAs were reacted in the presence of 2 ul of recombinase and4 ul of buffer at 25° C. for 12 hrs. A total of 20 ul of reactionmixture was used in this reaction. In order to detect the insertion ofgene cassette, PCR amplification was performed using primers forpolyhedrin locus amplification.

[0078] Baculo-Forward primer: 5-actgttttcgtaacagttttg-3 (SEQ. ID No.: 6)

[0079] Baculo-Reverse primer: 5-acaacgcacagaatctagc-3 (SEQ. ID No.: 7)

[0080] As the result, recombinant viral DNAs were generated with veryhigh efficiency. Meanwhile, liposome was prepared by mixing 5 ul of therecombinant reaction mixture with 5 ul of 10% lipofectin dilates, whichin turn was transferred to 50,000 of Sf21 cells in 96-well plate (SPL)followed by the incubation of the cells for 4 days. Protein expressionwas assayed after 3-day incubation of 50,000 of the Sf21 cells that wereinfected again with 10 ul of viruses. As for GFP viruses, the expressionof green fluorescence was detected under the fluorescence microscope.And, the exhibition of symptoms of GFP viral infection was detectedalso. The cells were isolated from each of the wells with SDS-PAGE, andthe expression of GFP was assayed using GFP-antibody with western blot.With regard to GFP proteins, the measured value showed similar patternas detected under the fluorescence microscope (see FIG. 22).

Example 10 High-Throughput Preparation and Enzyme Activity Screening ofLacZ Gene Mutants in 96-Well Plate Using GFP Recombinant Baculoviruses

[0081] In order to prepare desired recombinant baculoviruses (sizedabout 1˜3.2 kb) from multi-well plate simultaneously, the vBacHTS_GFPviral DNAs were treated with Bsu36I restriction enzyme. Gene cassettesof pEntr_LacZdel were employed in this study. The gene cassettes wereprepared in 96-well plate by PCR cloning of the gene of β-galactosidasefrom E. coli (sized about 3.2 kb), wherein the gene was deleted of3′-terminus. More specifically, pEntr_LacZ was prepared by PCR cloningof gene of LacZ. Afterward, the pEntr_LacZ was treated with ExoIII/S1deletion kit (#K0421) (from Fermentas) to generate of pEntr_LacZdel. Allthe processes were carried out in multi-well plate, while therecombination reaction, insect cell incubation and viral infection wereperformed with 8-channel pipette. 50 ng of gene cassettes and 200 ng ofvBacHTS baculoviral DNAs were reacted in the presence of 2 ul ofrecombines and 4 ul of buffer at 25° C. for 12 hrs. A total of 20 ul ofreaction mixture was used in this reaction. In order to detect theinsertion of gene cassette, PCR amplification was performed using a pairof primers (actgttttcgtaacagttttg and acaacgcacagaatctagc) for theamplification of polyhedrin locus. As the result, recombinant viruseswere generated at very high efficiency (see FIG. 25). Meanwhile,liposome was prepared by mixing 5 ul of the recombinant reaction mixtureand 5 ul of 10% lipofectin dilates, which in turn was transferred to50,000 of Sf21 cells in 96-well plate (SPL) followed by incubation ofthe cells for 4 days (see FIG. 26). In order to assay the expression ofGFP fusion proteins, SDS-PAGE analysis and western blotting using GFPantibodies were carried out (see FIG. 26).

[0082] In order to screen the gene activities, 1.5 ul of X-Gal (25 mg/mlin DMSO) of developer of β-galactosidase was added to each well thatcontained viruses incubated for 3 days after the primary infection.After 12 hrs of reaction, viruses containing β-galactosidase activitywere detected in 2 (two) wells (see FIG. 27).

Example 11 High-Throughput Heterologous Gene Expression UsingRecombinant Baculoviruses in 96-Well Plate and the Screening of EnzymeActivity Using the Same

[0083]S. cerevisie, which includes 124 protein kinase genes, wasemployed in this study. PCR amplification and cloning were carried outusing gene-specific primers to obtain 112 gene cassettes from the S.cerevisie genome. Recombinant baculoviruses were prepared by therecombination of the gene cassettes and vBacHTS_GFP vector DNA. Therespective recombinant viruses represented particular fluorescenceaccording to the proteins fused to GFP (see FIG. 28). Also, therecombinant baculoviruses having 32 human cDNA were prepared in the samemanner.

[0084] Firstly, cells were infected with these viruses and incubated for3 days. The cell lysate was generated using a buffer to lyse cells.Viruses, which exhibited protein kinase activities to substrates ofHiston H1, MBP (myelin basic protein) and Rb, were screened. 2 ug ofprotein substrates (histon H1, MBP and Rb) were reacted with 10 ul ofcell extracts at 30° C. for 10 min, in the presence of 10 uM ATP, 0.2uCi of P32-δ-labeled ATP (gamma, P32 ATP), and 2 ul of phosphate buffer[200 mM Tris-HCl (pH=8.0), 100 mM of MgCl₂, 10 mM of EGTA and 10 mM ofDTT]. After stopping the reaction by adding 120 ul of 1% phosphoric acidsolution, the reaction mixture was transferred to PVDF membrane (fromMilipore, #MAIP-N45). Afterward, the membrane was washed with detergent[10 mM Tris-HCl (pH-8.0), 1 mM EDTA and 150 mM NaCl] four times anddried, which in turn was exposed to phosphoscreen at room temperature,and then signals were detected with Phosphoimager (from MD or Fuji). Asthe result, it was observed that protein kinase activities wereincreased in some viruses (see FIG. 29). Subsequently, SDS-PAGEelectophoretic analysis was carried out to confirm the specificity ofprotein phosphorylation. At that time, the level of Rb proteinphosphorylation with regard to the concentration of protein kinaseincluded in cell extracts was detected, while varying the concentrationsof cell extracts. As the result, only the cell extracts of thebaculoviruses expressing plate#2-D3 protein and plate#2-A9 proteinincreased Rb protein phosphorylation in a concentration dependent manner(see FIG. 30). Meanwhile, proteins of plate#2-D1 and plate#2-D2exhibited very weak phosphorylation activities. And, it was acknowledgedthat the positive signals from the proteins of plate#2-D1 and plate#2-D2were resulted from auto-phosphorylation activities rather than from Rbprotein phosphorylation. Rb protein phosphorylations by plate#2-D3 andplate#2-A9 proteins exhibited a typical protein phosphorylation patternin that phosphorylation of Rb protein was dependent on theconcentrations of the substrate and the enzyme (FIG. 31). Thus, it ispossible to find out new useful proteins from baculoviral librariesobtained from the high-throughput system of the present inventionaccording to the present invention.

Example 12 The Preparation of pBacHTS2 and pBacHTS2 GFP

[0085] Baculoviral transfer vector of pBacHTS2 was prepared in order togenerate vBacHTS2 viruses (SEQ. ID No.8: the sequences of its polyhedrinlocus) that can replicate and can be applied to recombination reactionin vitro. The pBacHTS2 had BAC vector originated replication origin andCmR (chloramphenichol resistance gene) at Bsu36I restriction enzymerecognition site located between attR1 site and attR2 site thereof.Thus, it was a kind of BAC vectors that can replicate in bacteria.Replacing the BAC vector originated replication origin and CmR withdesired gene cassette generated vBacHTS viruses in vitro.

[0086] In order to prepare pBacHTS2, firstly, 6.5 kb of DNA fragmentcontaining BAC (Bacterial artificial chromosome) vector replicationorigin and CmR was generated using pBACe3.6 (Genbank:U80929) as atemplate. Then, PCR amplification was carried out using 10 pmole of apair of sequences having Bsu36I recognition sequences, 200 uM of dNTPsand 2.5 U of Pfu turbo polymerase (Stratagene) with PCR cycler (AppliedBiosystem, Gene Amp PCR System 2700). A total of 50 ul of reactionmixture was used. PCR was carried out for 20 cycles (DNA denaturation at95° C., 30 sec, DNA extension at 60° C., 30 sec, DNA amplification at72° C., 7 min). PCR products of 6.5 kb of DNA fragment and vBacHTSvector were digested by Bsu36I, and were reacted in the presence of T4ligase at 16° C. overnight. E. coli (DH5a) was transformed using theresulting mixture, which in turn was incubated with Cm and Amp medium(for selection) to produce pBacHTS2. Likewise, Bsu36I treated PCRproducts of 6.5 kb of DNA fragment were inserted to Bsu36I site of thepBacHTS_GFP vector so as to obtain pBacHTS2_GFP vector.

Example 13 Preparation of Viruses Having Site-Specific RecombinationSites

[0087] Baculoviruses were prepared using homologous recombination, whichreplicated in bacteria. Briefly, the homologous recombination wasinduced in Sf21 cells by transferring a mixture of pBacHTS2 plasmid,Bsu36I digested BacPAK6 DNA and Lipofectin (from LTI). The infectedcells were incubated at 27° C. for 4 days, and were transferred to 5×10⁶of Sf21 cells (in 100 mm of dish). Thereby, high titer of viruses wasobtained. Then, precipitates of viral particles were produced bycentrifugation after adding 2 ml of 50% PEG6000 to the cell culturemedium. Next, proteinase K was added to the mixture at 42° C. and leftthe mixture alone for 2 hrs to dismantle viral envelope. The virallysate was extracted twice using the same amount of phenol/chloroform,and ethanol was added thereto. Thereby, precipitated and purifiedvBacHTS2 viral DNAs (SEQ. ID No.8: the sequences of its polyhedrinlocus) were obtained. Then, cell lines (from LTI) were transformed withthe purified DNAs using MicroPulser (from Bio-Rad), which are in turnincubated in Cm medium to form bacterial colony. The bacteria wereincubated in 1 liter of 2×YT medium (10 g of Yeast extract powder, 16 gof tryptone, 5 g of NaCl within per 1 (one) Liter), then DNAs werepurified and named as bBacHTS2 (FIG. 16). Thus, according to thismethod, it is possible to reduce the required time-period and costsignificantly, since the viral DNAs can be obtained from bacterialculture without the need of incubation in insect cells. Purified 10 ugof DNAs was digested with 50 unit of Bsu36I at 37° C. for 5 hrs, whichin turn was heat treated at 80° C. for 20 min to inactivate Bsu361enzyme. Likewise, vBacHTS2_GFP (SEQ. ID No.:9) was prepared frompBacHTS2_GFP vector by homologous recombination; and then wastransferred to DH10B by electrophoration. The DNAs were purified andnamed as bBacHTS2_GFP (see FIG. 16).

Example 14 Preparation of the Desired Recombinant Viruses Using vBacHTS2and vBacHTS2 GFP DNAs Generated from Bacteria

[0088] 50 ng of the pEntr_GFP and the pEntr_Gus gene cassettes werereacted with 200 ng of Bsu36I treated vBacHTS2 viral DNAs in thepresence of 1 ul of integrase mixture in vitro at 25° C. for 12 hrs. Thesame reaction was carried out except for the use of the pEntr_Gus genecassette instead of pEntr_GFP gene cassette. PCR was performed asdisclosed in the Examples mentioned above. PCR amplification was carriedout using the reaction mixture, as templates, and a pair of primersamplifying viral polyhedrin locus to confirm whether recombinationreaction occurred. A total of 20 cycles of PCR were carried out usingeach of 10 pmole of the primers and 1 tube of Bioneer premix kit, inwhich one cycle of PCR was performed at 95° C. for 30 sec, 50° C. for 30see and 72° C. for 3 min. After completing PCR reaction, electrophoresiswas carried out for the PCR products on 1% agarose gel to confirmefficient performance of recombination. The gene cassette reactionmixture was transferred to Sf21 cells together with 5 ul of Lipofectin(from LTI) so as to obtain baculoviruses.

[0089] Green fluorescence was observed in insect cells, after 2 daysfrom transferring GFP gene cassette reaction mixture thereto. Inaddition, most of the cells represented green fluorescence after 4 daysfrom transfer (see FIG. 25). Sf21 cells were infected again with theviral culture medium to confirm green fluorescence and the symptoms ofinfection. Likewise, pEntr_Gus gene cassette was reacted with Bsu36Itreated vBacHTS2 DNAs at 25° C. for 12 hrs. Then, the obtained reactionmixture was transferred to Sf21 cells and was incubated at 27° C. for 4days. When 6 ul of X-Gluc (20 mg/ml in DMSO) was added to incubatedcells, all the culture medium represented strong blue, which supportednot only the expressions of Gus genes but also efficient generation ofrecombinant viruses containing Gus genes.

1 9 1 10000 DNA Artificial Sequence Polyhedrin locus of vBacHTS virus 1gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa tgacatcatt tgtataatga 60catcatcccc tgattgtgtt ttacaagtag aattctatcc gtaaagcgag ttcagttttg 120aaaacaaatg agtcatacct aaacacgtta ataatcttct gatatcagct tatgactcaa 180gttatgagcc gtgtgcaaaa catgagataa gtttatgaca tcatccactg atcgtgcgtt 240acaagtagaa ttctactcgt aaagccagtt cggttatgag ccgtgtgcaa aacatgacat 300cagcttatga ctcatacttg attgtgtttt acgcgtagaa ttctactcgt aaagcgagtt 360cggttatgag ccgtgtgcaa aacatgacat cagcttatga gtcataatta atcgtgcgtt 420acaagtagaa ttctactcgt aaagcgagtt gaaggatcat atttagttgc gtttatgaga 480taagattgaa agcacgtgta aaatgtttcc cgcgcgttgg cacaactatt tacaatgcgg 540ccaagttata aaagattcta atctgatatg ttttaaaaca cctttgcggc ccgagttgtt 600tgcgtacgtg actagcgaag aagatgtgtg gaccgcagaa cagatagtaa aacaaaaccc 660tagtattgga gcaataatcg atttaaccaa cacgtctaaa tattatgatg gtgtgcattt 720tttgcgggcg ggcctgttat acaaaaaaat tcaagtacct ggccagactt tgccgcctga 780aagcatagtt caagaattta ttgacacggt aaaagaattt acagaaaagt gtcccggcat 840gttggtgggc gtgcactgca cacacggtat taatcgcacc ggttacatgg tgtgcagata 900tttaatgcac accctgggta ttgcgccgca ggaagccata gatagattcg aaaaagccag 960aggtcacaaa attgaaagac aaaattacgt tcaagattta ttaatttaat taatattatt 1020tgcattcttt aacaaatact ttatcctatt ttcaaattgt tgcgcttctt ccagcgaacc 1080aaaactatgc ttcgcttgct ccgtttagct tgtagccgat cagtggcgtt gttccaatcg 1140acggtaggat taggccggat attctccacc acaatgttgg caacgttgat gttacgttta 1200tgcttttggt tttccacgta cgtcttttgg ccggtaatag ccgtaaacgt agtgccgtcg 1260cgcgtcacgc acaacaccgg atgtttgcgc ttgtccgcgg ggtattgaac cgcgcgatcc 1320gacaaatcca ccactttggc aactaaatcg gtgacctgcg cgtctttttt ctgcattatt 1380tcgtctttct tttgcatggt ttcctggaag ccggtgtaca tgcggtttag atcagtcatg 1440acgcgcgtga cctgcaaatc tttggcctcg atctgcttgt ccttgatggc aacgatgcgt 1500tcaataaact cttgtttttt aacaagttcc tcggtttttt gcgccaccac cgcttgcagc 1560gcgtttgtgt gctcggtgaa tgtcgcaatc agcttagtca ccaactgttt gctctcctcc 1620tcccgttgtt tgatcgcggg atcgtacttg ccggtgcaga gcacttgagg aattacttct 1680tctaaaagcc attcttgtaa ttctatggcg taaggcaatt tggacttcat aatcagctga 1740atcacgccgg atttagtaat gagcactgta tgcggctgca aatacagcgg gtcgcccctt 1800ttcacgacgc tgttagaggt agggccccca ttttggatgg tctgctcaaa taacgatttg 1860tatttattgt ctacatgaac acgtatagct ttatcacaaa ctgtatattt taaactgtta 1920gcgacgtcct tggccacgaa ccggacctgt tggtcgcgct ctagcacgta ccgcaggttg 1980aacgtatctt ctccaaattt aaattctcca attttaacgc gagccatttt gatacacgtg 2040tgtcgatttt gcaacaacta ttgtttttta acgcaaacta aacttattgt ggtaagcaat 2100aattaaatat gggggaacat gcgccgctac aacactcgtc gttatgaacg cagacggcgc 2160cggtctcggc gcaagcggct aaaacgtgtt gcgcgttcaa cgcggcaaac atcgcaaaag 2220ccaatagtac agttttgatt tgcatattaa cggcgatttt ttaaattatc ttatttaata 2280aatagttatg acgcctacaa ctccccgccc gcgttgactc gctgcacctc gagcagttcg 2340ttgacgcctt cctccgtgtg gccgaacacg tcgagcgggt ggtcgatgac cagcggcgtg 2400ccgcacgcga cgcacaagta tctgtacacc gaatgatcgt cgggcgaagg cacgtcggcc 2460tccaagtggc aatattggca aattcgaaaa tatatacagt tgggttgttt gcgcatatct 2520atcgtggcgt tgggcatgta cgtccgaacg ttgatttgca tgcaagccga aattaaatca 2580ttgcgattag tgcgattaaa acgttgtaca tcctcgcttt taatcatgcc gtcgattaaa 2640tcgcgcaatc gagtcaagtg atcaaagtgt ggaataatgt tttctttgta ttcccgagtc 2700aagcgcagcg cgtattttaa caaactagcc atcttgtaag ttagtttcat ttaatgcaac 2760tttatccaat aatatattat gtatcgcacg tcaagaatta acaatgcgcc cgttgtcgca 2820tctcaacacg actatgatag agatcaaata aagcgcgaat taaatagctt gcgacgcaac 2880gtgcacgatc tgtgcacgcg ttccggcacg agctttgatt gtaataagtt tttacgaagc 2940gatgacatga cccccgtagt gacaacgatc acgcccaaaa gaactgccga ctacaaaatt 3000accgagtatg tcggtgacgt taaaactatt aagccatcca atcgaccgtt agtcgaatca 3060ggaccgctgg tgcgagaagc cgcgaagtat ggcgaatgca tcgtataacg tgtggagtcc 3120gctcattaga gcgtcatgtt tagacaagaa agctacatat ttaattgatc ccgatgattt 3180tattgataaa ttgaccctaa ctccatacac ggtattctac aatggcgggg ttttggtcaa 3240aatttccgga ctgcgattgt acatgctgtt aacggctccg cccactatta atgaaattaa 3300aaattccaat tttaaaaaac gcagcaagag aaacatttgt atgaaagaat gcgtagaagg 3360aaagaaaaat gtcgtcgaca tgctgaacaa caagattaat atgcctccgt gtataaaaaa 3420aatattgaac gatttgaaag aaaacaatgt accgcgcggc ggtatgtaca ggaagaggtt 3480tatactaaac tgttacattg caaacgtggt ttcgtgtgcc aagtgtgaaa accgatgttt 3540aatcaaggct ctgacgcatt tctacaacca cgactccaag tgtgtgggtg aagtcatgca 3600tcttttaatc aaatcccaag atgtgtataa accaccaaac tgccaaaaaa tgaaaactgt 3660cgacaagctc tgtccgtttg ctggcaactg caagggtctc aatcctattt gtaattattg 3720aataataaaa caattataaa tgctaaattt gttttttatt aacgatacaa accaaacgca 3780acaagaacat ttgtagtatt atctataatt gaaaacgcgt agttataatc gctgaggtaa 3840tatttaaaat cattttcaaa tgattcacag ttaatttgcg acaatataat tttattttca 3900cataaactag acgccttgtc gtcttcttct tcgtattcct tctctttttc atttttctcc 3960tcataaaaat taacatagtt attatcgtat ccatatatgt atctatcgta tagagtaaat 4020tttttgttgt cataaatata tatgtctttt ttaatggggt gtatagtacc gctgcgcata 4080gtttttctgt aatttacaac agtgctattt tctggtagtt cttcggagtg tgttgcttta 4140attattaaat ttatataatc aatgaatttg ggatcgtcgg ttttgtacaa tatgttgccg 4200gcatagtacg cagcttcttc tagttcaatt acaccatttt ttagcagcac cggattaaca 4260taactttcca aaatgttgta cgaaccgtta aacaaaaaca gttcacctcc cttttctata 4320ctattgtctg cgagcagttg tttgttgtta aaaataacag ccattgtaat gagacgcaca 4380aactaatatc acaaactgga aatgtctatc aatatatagt tgctgatatc atggagataa 4440ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt 4500aataaaaaaa cctataaata cggatcccgg ggtaccacaa gtttgtacaa aaaagctgaa 4560cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag 4620actacataat actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc 4680cttaggaatt cgcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg 4740cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtgtgcttct 4800agaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat 4860gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca 4920cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa 4980agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa 5040gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg 5100ggaatataga attcctcagg tcgaccatag tgactggata tgttgtgttt tacagtatta 5160tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt 5220ttctcgttca gctttcttgt acaaagtggt ctcgagttaa ttaattgatc cgggttatta 5280gtacatttat taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta 5340ttttaataat tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg 5400attttcagcg tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag 5460gtttcgatta gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg 5520attttcgctc aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat 5580attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt 5640tgtatttctt tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaataa 5700agcttggaca tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc 5760ccaaccctcg tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct 5820attaattgtg tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttggaattat 5880ttctgattgc gggcgttttt gggcgggttt caatctaact gtgcccgatt ttaattcaga 5940caacacgtta gaaagcgatg gtgcaggcgg tggtaacatt tcagacggca aatctactaa 6000tggcggcggt ggtggagctg atgataaatc taccatcggt ggaggcgcag gcggggctgg 6060cggcggaggc ggaggcggag gtggtggcgg tgatgcagac ggcggtttag gctcaaatgt 6120ctctttaggc aacacagtcg gcacctcaac tattgtactg gtttcgggcg ccgtttttgg 6180tttgaccggt ctgagacgag tgcgattttt ttcgtttcta atagcttcca acaattgttg 6240tctgtcgtct aaaggtgcag cgggttgagg ttccgtcggc attggtggag cgggcggcaa 6300ttcagacatc gatggtggtg gtggtggtgg aggcgctgga atgttaggca cgggagaagg 6360tggtggcggc ggtgccgccg gtataatttg ttctggttta gtttgttcgc gcacgattgt 6420gggcaccggc gcaggcgccg ctggctgcac aacggaaggt cgtctgcttc gaggcagcgc 6480ttggggtggt ggcaattcaa tattataatt ggaatacaaa tcgtaaaaat ctgctataag 6540cattgtaatt tcgctatcgt ttaccgtgcc gatatttaac aaccgctcaa tgtaagcaat 6600tgtattgtaa agagattgtc tcaagctcgg atcccgcacg ccgataacaa gccttttcat 6660ttttactaca gcattgtagt ggcgagacac ttcgctgtcg tcgacgtaca tgtatgcttt 6720gttgtcaaaa acgtcgttgg caagctttaa aatatttaaa agaacatctc tgttcagcac 6780cactgtgttg tcgtaaatgt tgtttttgat aatttgcgct tccgcagtat cgacacgttc 6840aaaaaattga tgcgcatcaa ttttgttgtt cctattattg aataaataag attgtacaga 6900ttcatatcta cgattcgtca tggccaccac aaatgctacg ctgcaaacgc tggtacaatt 6960ttacgaaaac tgcaaaaacg tcaaaactcg gtataaaata atcaacgggc gctttggcaa 7020aatatctatt ttatcgcaca agcccactag caaattgtat ttgcagaaaa caatttcggc 7080gcacaatttt aacgctgacg aaataaaagt tcaccagtta atgagcgacc acccaaattt 7140tataaaaatc tattttaatc acggttccat caacaaccaa gtgatcgtga tggactacat 7200tgactgtccc gatttatttg aaacactaca aattaaaggc gagctttcgt accaacttgt 7260tagcaatatt attagacagc tgtgtgaagc gctcaacgat ttgcacaagc acaatttcat 7320acacaacgac ataaaactcg aaaatgtctt atatttcgaa gcacttgatc gcgtgtatgt 7380ttgcgattac ggattgtgca aacacgaaaa ctcacttagc gtgcacgacg gcacgttgga 7440gtattttagt ccggaaaaaa ttcgacacac aactatgcac gtttcgtttg actggtacgc 7500cgtcggcgtg ttaacataca agttgctaac cggcggccga cacccatttg aaaaaagcga 7560agacgaaatg ttggacttga atagcatgaa gcgtcgtcag caatacaatg acattggcgt 7620tttaaaacac gttcgtaacg ttaacgctcg tgactttgtg tactgcctaa caagatacaa 7680catagattgt agactcacaa attacaaaca aattataaaa catgagtttt tgtcgtaaaa 7740atgccacttg ttttacgagt agaattctac gtgtaacaca cgatctaaaa gatgatgtca 7800ttttttatca atgactcatt tgttttaaaa cagacttgtt ttacgagtag aattctacgt 7860gtaaagcatg atcgtgagtg gtgttaataa aatcataaaa attattgtaa atgtttatta 7920tttaaaaacg attcaaatat ataataaaaa caatctacat ctatttcttc acaatccata 7980acacacaaca ggtccatcaa tgagtttttg tctttatccg acatactatg tgcatgtaac 8040aaatcaaata catcttttaa atttttatac acatctttac attgtctacc aaaatcttta 8100ataaccctat aacaaggaaa agacttttct tcttgcgtgg ttttgccgcg cagatattga 8160aataaaatgt gcatgcacga caacttgtgt ttactaaaat gctccttgcc tataccgcaa 8220aaccggccat acatttcggc gattacacgc ggacaattgt acgattcgtc tacgtgtaaa 8280cgatcatcat aatcactctt gcgcaaacga ataaattttt tcaccgcttc cgacaaacga 8340ggcaccaatt cggcgggcac gcttcgatac attattctgt gcacataagt taccacacaa 8400aatttattgt accaccatcc gacaacgtcg ttattagggt tgaacacgtt ggcgatgcgc 8460agcagtttcc cgtttctcat gaaatattca aagcggccca aaataatttg caagcaatcc 8520aacatgtctt gagaaatttc tcgttcaaaa ttgttcaaag agaatatctg ccatccgttt 8580tgaacgcgca cgctgacggg aaccaccgca tcgatttgct ccaacacttc acggacgtta 8640tcgtcgatgc ccatcgtttc gctggtgctg aaccaatggg aaaggctctt gatggaatcg 8700cccgcgtcta tcatcttgac cgcttcgtca aaggtgcaac tgccgctctt caaacgccgc 8760atagcggtca cgtcccgctc tatgcacgac ataccgttta cgtacgattc tgataggtat 8820tcctgaacta tacggtaatg gtgatacgac tcgccataca cgtcgtgcac ctcattgtat 8880ttagcataat aattgtaaat tattaacttt gcagcgagag acatgttgtc agtaaagcgg 8940tgctaggctc aataatactg atgtacaggc acgcgtgcta tttatatata atttcgcaag 9000gaggggagct gttatcggtt gctattatta aagaatggcc gtctgttttt atcacaagct 9060tggcagcctc aaccatgaag cgtcgtcatt gtaaattaaa ttctctgcct caagaattat 9120ttgacaagat tgtcgagtat ttatctttat ctgattactg caatttggtg cttgtctgta 9180aaagaccttc tagtaaatat aacgtgatat ttgatagtac taatcaccaa catttgaaag 9240gcgtgtacaa aaagacagac gtgcaaataa caagctacaa cgaatacatc aactgtattt 9300gcaacgaact gagacaagac gaattctatg ccaaatcatc atggattgcg agtatttgcg 9360gtcaccagag agcgacaatt tttagtgtaa caaataaaca agtagaaatg aaatatcatt 9420tgtataatat agcaattgtg gaaagtgaag attgcaacgg attttaccca tttgagccaa 9480cgcgcgattg tttaatatgc aaacaaaaaa accaatgtcc tcgtaattca tttattgttt 9540cgttgtgtaa atatttagaa aaacaaaatg tacaatcaaa ctttatatat tatttatacg 9600aaataaatac ataataataa ctattataca tgtttttatt ttacaatact tcctgtataa 9660cctctctaac tacattagga gtacaatcca cgtcaattac acgtttagct atttttctaa 9720ttttgtaatg tttatcgtag agtttttcgt taatacattg aatagccaac aagggatttg 9780ggtgcacacc gtcatagagt acttccatgt cgtcttcaaa gcgcattttt cgcttgcgaa 9840aatgccgctc ttggcccaaa acaaaagcga gtttgatgcg gtcgtcgatg cgttccgaaa 9900atacggccaa atgctggtgt ttggtgatgt cgcgcggaaa cgtcaccgtg ccatttttgc 9960tttccgccac gacggcggtt ttcaattttt cggccgactg 10000 2 10000 DNA ArtificialSequence Polyhedrin locus of vBacHTS_His 2 gaattctacc cgtaaagcgagtttagtttt gaaaaacaaa tgacatcatt tgtataatga 60 catcatcccc tgattgtgttttacaagtag aattctatcc gtaaagcgag ttcagttttg 120 aaaacaaatg agtcatacctaaacacgtta ataatcttct gatatcagct tatgactcaa 180 gttatgagcc gtgtgcaaaacatgagataa gtttatgaca tcatccactg atcgtgcgtt 240 acaagtagaa ttctactcgtaaagccagtt cggttatgag ccgtgtgcaa aacatgacat 300 cagcttatga ctcatacttgattgtgtttt acgcgtagaa ttctactcgt aaagcgagtt 360 cggttatgag ccgtgtgcaaaacatgacat cagcttatga gtcataatta atcgtgcgtt 420 acaagtagaa ttctactcgtaaagcgagtt gaaggatcat atttagttgc gtttatgaga 480 taagattgaa agcacgtgtaaaatgtttcc cgcgcgttgg cacaactatt tacaatgcgg 540 ccaagttata aaagattctaatctgatatg ttttaaaaca cctttgcggc ccgagttgtt 600 tgcgtacgtg actagcgaagaagatgtgtg gaccgcagaa cagatagtaa aacaaaaccc 660 tagtattgga gcaataatcgatttaaccaa cacgtctaaa tattatgatg gtgtgcattt 720 tttgcgggcg ggcctgttatacaaaaaaat tcaagtacct ggccagactt tgccgcctga 780 aagcatagtt caagaatttattgacacggt aaaagaattt acagaaaagt gtcccggcat 840 gttggtgggc gtgcactgcacacacggtat taatcgcacc ggttacatgg tgtgcagata 900 tttaatgcac accctgggtattgcgccgca ggaagccata gatagattcg aaaaagccag 960 aggtcacaaa attgaaagacaaaattacgt tcaagattta ttaatttaat taatattatt 1020 tgcattcttt aacaaatactttatcctatt ttcaaattgt tgcgcttctt ccagcgaacc 1080 aaaactatgc ttcgcttgctccgtttagct tgtagccgat cagtggcgtt gttccaatcg 1140 acggtaggat taggccggatattctccacc acaatgttgg caacgttgat gttacgttta 1200 tgcttttggt tttccacgtacgtcttttgg ccggtaatag ccgtaaacgt agtgccgtcg 1260 cgcgtcacgc acaacaccggatgtttgcgc ttgtccgcgg ggtattgaac cgcgcgatcc 1320 gacaaatcca ccactttggcaactaaatcg gtgacctgcg cgtctttttt ctgcattatt 1380 tcgtctttct tttgcatggtttcctggaag ccggtgtaca tgcggtttag atcagtcatg 1440 acgcgcgtga cctgcaaatctttggcctcg atctgcttgt ccttgatggc aacgatgcgt 1500 tcaataaact cttgttttttaacaagttcc tcggtttttt gcgccaccac cgcttgcagc 1560 gcgtttgtgt gctcggtgaatgtcgcaatc agcttagtca ccaactgttt gctctcctcc 1620 tcccgttgtt tgatcgcgggatcgtacttg ccggtgcaga gcacttgagg aattacttct 1680 tctaaaagcc attcttgtaattctatggcg taaggcaatt tggacttcat aatcagctga 1740 atcacgccgg atttagtaatgagcactgta tgcggctgca aatacagcgg gtcgcccctt 1800 ttcacgacgc tgttagaggtagggccccca ttttggatgg tctgctcaaa taacgatttg 1860 tatttattgt ctacatgaacacgtatagct ttatcacaaa ctgtatattt taaactgtta 1920 gcgacgtcct tggccacgaaccggacctgt tggtcgcgct ctagcacgta ccgcaggttg 1980 aacgtatctt ctccaaatttaaattctcca attttaacgc gagccatttt gatacacgtg 2040 tgtcgatttt gcaacaactattgtttttta acgcaaacta aacttattgt ggtaagcaat 2100 aattaaatat gggggaacatgcgccgctac aacactcgtc gttatgaacg cagacggcgc 2160 cggtctcggc gcaagcggctaaaacgtgtt gcgcgttcaa cgcggcaaac atcgcaaaag 2220 ccaatagtac agttttgatttgcatattaa cggcgatttt ttaaattatc ttatttaata 2280 aatagttatg acgcctacaactccccgccc gcgttgactc gctgcacctc gagcagttcg 2340 ttgacgcctt cctccgtgtggccgaacacg tcgagcgggt ggtcgatgac cagcggcgtg 2400 ccgcacgcga cgcacaagtatctgtacacc gaatgatcgt cgggcgaagg cacgtcggcc 2460 tccaagtggc aatattggcaaattcgaaaa tatatacagt tgggttgttt gcgcatatct 2520 atcgtggcgt tgggcatgtacgtccgaacg ttgatttgca tgcaagccga aattaaatca 2580 ttgcgattag tgcgattaaaacgttgtaca tcctcgcttt taatcatgcc gtcgattaaa 2640 tcgcgcaatc gagtcaagtgatcaaagtgt ggaataatgt tttctttgta ttcccgagtc 2700 aagcgcagcg cgtattttaacaaactagcc atcttgtaag ttagtttcat ttaatgcaac 2760 tttatccaat aatatattatgtatcgcacg tcaagaatta acaatgcgcc cgttgtcgca 2820 tctcaacacg actatgatagagatcaaata aagcgcgaat taaatagctt gcgacgcaac 2880 gtgcacgatc tgtgcacgcgttccggcacg agctttgatt gtaataagtt tttacgaagc 2940 gatgacatga cccccgtagtgacaacgatc acgcccaaaa gaactgccga ctacaaaatt 3000 accgagtatg tcggtgacgttaaaactatt aagccatcca atcgaccgtt agtcgaatca 3060 ggaccgctgg tgcgagaagccgcgaagtat ggcgaatgca tcgtataacg tgtggagtcc 3120 gctcattaga gcgtcatgtttagacaagaa agctacatat ttaattgatc ccgatgattt 3180 tattgataaa ttgaccctaactccatacac ggtattctac aatggcgggg ttttggtcaa 3240 aatttccgga ctgcgattgtacatgctgtt aacggctccg cccactatta atgaaattaa 3300 aaattccaat tttaaaaaacgcagcaagag aaacatttgt atgaaagaat gcgtagaagg 3360 aaagaaaaat gtcgtcgacatgctgaacaa caagattaat atgcctccgt gtataaaaaa 3420 aatattgaac gatttgaaagaaaacaatgt accgcgcggc ggtatgtaca ggaagaggtt 3480 tatactaaac tgttacattgcaaacgtggt ttcgtgtgcc aagtgtgaaa accgatgttt 3540 aatcaaggct ctgacgcatttctacaacca cgactccaag tgtgtgggtg aagtcatgca 3600 tcttttaatc aaatcccaagatgtgtataa accaccaaac tgccaaaaaa tgaaaactgt 3660 cgacaagctc tgtccgtttgctggcaactg caagggtctc aatcctattt gtaattattg 3720 aataataaaa caattataaatgctaaattt gttttttatt aacgatacaa accaaacgca 3780 acaagaacat ttgtagtattatctataatt gaaaacgcgt agttataatc gctgaggtaa 3840 tatttaaaat cattttcaaatgattcacag ttaatttgcg acaatataat tttattttca 3900 cataaactag acgccttgtcgtcttcttct tcgtattcct tctctttttc atttttctcc 3960 tcataaaaat taacatagttattatcgtat ccatatatgt atctatcgta tagagtaaat 4020 tttttgttgt cataaatatatatgtctttt ttaatggggt gtatagtacc gctgcgcata 4080 gtttttctgt aatttacaacagtgctattt tctggtagtt cttcggagtg tgttgcttta 4140 attattaaat ttatataatcaatgaatttg ggatcgtcgg ttttgtacaa tatgttgccg 4200 gcatagtacg cagcttcttctagttcaatt acaccatttt ttagcagcac cggattaaca 4260 taactttcca aaatgttgtacgaaccgtta aacaaaaaca gttcacctcc cttttctata 4320 ctattgtctg cgagcagttgtttgttgtta aaaataacag ccattgtaat gagacgcaca 4380 aactaatatc acaaactggaaatgtctatc aatatatagt tgctgatatc atggagataa 4440 ttaaaatgat aaccatctcgcaaataaata agtattttac tgttttcgta acagttttgt 4500 aataaaaaaa cctataaatacggatctatg catcatcacc atcatcacgg atcccggggt 4560 accacaagtt tgtacaaaaaagctgaacga gaaacgtaaa atgatataaa tatcaatata 4620 ttaaattaga ttttgcataaaaaacagact acataatact gtaaaacaca acatatccag 4680 tcactatggc ggccgcattaggcacccctt aggaattcgc ttactaaaag ccagataaca 4740 gtatgcgtat ttgcgcgctgatttttgcgg tataagaata tatactgata tgtatacccg 4800 aagtatgtca aaaagaggtgtgcttctaga atgcagttta aggtttacac ctataaaaga 4860 gagagccgtt atcgtctgtttgtggatgta cagagtgata ttattgacac gcccgggcga 4920 cggatggtga tccccctggccagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt 4980 tacccggtgg tgcatatcggggatgaaagc tggcgcatga tgaccaccga tatggccagt 5040 gtgccggtct ccgttatcggggaagaagtg gctgatctca gccaccgcga aaatgacatc 5100 aaaaacgcca ttaacctgatgttctgggga atatagaatt cctcaggtcg accatagtga 5160 ctggatatgt tgtgttttacagtattatgt agtctgtttt ttatgcaaaa tctaatttaa 5220 tatattgata tttatatcattttacgtttc tcgttcagct ttcttgtaca aagtggtctc 5280 gagttaatta attgatccgggttattagta catttattaa gcgctagatt ctgtgcgttg 5340 ttgatttaca gacaattgttgtacgtattt taataattca ttaaatttat aatctttagg 5400 gtggtatgtt agagcgaaaatcaaatgatt ttcagcgtct ttatatctga atttaaatat 5460 taaatcctca atagatttgtaaaataggtt tcgattagtt tcaaacaagg gttgtttttc 5520 cgaaccgatg gctggactatctaatggatt ttcgctcaac gccacaaaac ttgccaaatc 5580 ttgtagcagc aatctagctttgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc 5640 gacgtcgttc aaaatattatgcgcttttgt atttctttca tcactgtcgt tagtgtacaa 5700 ttgactcgac gtaaacacgttaaataaagc ttggacatat ttaacatcgg gcgtgttagc 5760 tttattaggc cgattatcgtcgtcgtccca accctcgtcg ttagaagttg cttccgaaga 5820 cgattttgcc atagccacacgacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa 5880 atttgtagtt gagctttttggaattatttc tgattgcggg cgtttttggg cgggtttcaa 5940 tctaactgtg cccgattttaattcagacaa cacgttagaa agcgatggtg caggcggtgg 6000 taacatttca gacggcaaatctactaatgg cggcggtggt ggagctgatg ataaatctac 6060 catcggtgga ggcgcaggcggggctggcgg cggaggcgga ggcggaggtg gtggcggtga 6120 tgcagacggc ggtttaggctcaaatgtctc tttaggcaac acagtcggca cctcaactat 6180 tgtactggtt tcgggcgccgtttttggttt gaccggtctg agacgagtgc gatttttttc 6240 gtttctaata gcttccaacaattgttgtct gtcgtctaaa ggtgcagcgg gttgaggttc 6300 cgtcggcatt ggtggagcgggcggcaattc agacatcgat ggtggtggtg gtggtggagg 6360 cgctggaatg ttaggcacgggagaaggtgg tggcggcggt gccgccggta taatttgttc 6420 tggtttagtt tgttcgcgcacgattgtggg caccggcgca ggcgccgctg gctgcacaac 6480 ggaaggtcgt ctgcttcgaggcagcgcttg gggtggtggc aattcaatat tataattgga 6540 atacaaatcg taaaaatctgctataagcat tgtaatttcg ctatcgttta ccgtgccgat 6600 atttaacaac cgctcaatgtaagcaattgt attgtaaaga gattgtctca agctcggatc 6660 ccgcacgccg ataacaagccttttcatttt tactacagca ttgtagtggc gagacacttc 6720 gctgtcgtcg acgtacatgtatgctttgtt gtcaaaaacg tcgttggcaa gctttaaaat 6780 atttaaaaga acatctctgttcagcaccac tgtgttgtcg taaatgttgt ttttgataat 6840 ttgcgcttcc gcagtatcgacacgttcaaa aaattgatgc gcatcaattt tgttgttcct 6900 attattgaat aaataagattgtacagattc atatctacga ttcgtcatgg ccaccacaaa 6960 tgctacgctg caaacgctggtacaatttta cgaaaactgc aaaaacgtca aaactcggta 7020 taaaataatc aacgggcgctttggcaaaat atctatttta tcgcacaagc ccactagcaa 7080 attgtatttg cagaaaacaatttcggcgca caattttaac gctgacgaaa taaaagttca 7140 ccagttaatg agcgaccacccaaattttat aaaaatctat tttaatcacg gttccatcaa 7200 caaccaagtg atcgtgatggactacattga ctgtcccgat ttatttgaaa cactacaaat 7260 taaaggcgag ctttcgtaccaacttgttag caatattatt agacagctgt gtgaagcgct 7320 caacgatttg cacaagcacaatttcataca caacgacata aaactcgaaa atgtcttata 7380 tttcgaagca cttgatcgcgtgtatgtttg cgattacgga ttgtgcaaac acgaaaactc 7440 acttagcgtg cacgacggcacgttggagta ttttagtccg gaaaaaattc gacacacaac 7500 tatgcacgtt tcgtttgactggtacgccgt cggcgtgtta acatacaagt tgctaaccgg 7560 cggccgacac ccatttgaaaaaagcgaaga cgaaatgttg gacttgaata gcatgaagcg 7620 tcgtcagcaa tacaatgacattggcgtttt aaaacacgtt cgtaacgtta acgctcgtga 7680 ctttgtgtac tgcctaacaagatacaacat agattgtaga ctcacaaatt acaaacaaat 7740 tataaaacat gagtttttgtcgtaaaaatg ccacttgttt tacgagtaga attctacgtg 7800 taacacacga tctaaaagatgatgtcattt tttatcaatg actcatttgt tttaaaacag 7860 acttgtttta cgagtagaattctacgtgta aagcatgatc gtgagtggtg ttaataaaat 7920 cataaaaatt attgtaaatgtttattattt aaaaacgatt caaatatata ataaaaacaa 7980 tctacatcta tttcttcacaatccataaca cacaacaggt ccatcaatga gtttttgtct 8040 ttatccgaca tactatgtgcatgtaacaaa tcaaatacat cttttaaatt tttatacaca 8100 tctttacatt gtctaccaaaatctttaata accctataac aaggaaaaga cttttcttct 8160 tgcgtggttt tgccgcgcagatattgaaat aaaatgtgca tgcacgacaa cttgtgttta 8220 ctaaaatgct ccttgcctataccgcaaaac cggccataca tttcggcgat tacacgcgga 8280 caattgtacg attcgtctacgtgtaaacga tcatcataat cactcttgcg caaacgaata 8340 aattttttca ccgcttccgacaaacgaggc accaattcgg cgggcacgct tcgatacatt 8400 attctgtgca cataagttaccacacaaaat ttattgtacc accatccgac aacgtcgtta 8460 ttagggttga acacgttggcgatgcgcagc agtttcccgt ttctcatgaa atattcaaag 8520 cggcccaaaa taatttgcaagcaatccaac atgtcttgag aaatttctcg ttcaaaattg 8580 ttcaaagaga atatctgccatccgttttga acgcgcacgc tgacgggaac caccgcatcg 8640 atttgctcca acacttcacggacgttatcg tcgatgccca tcgtttcgct ggtgctgaac 8700 caatgggaaa ggctcttgatggaatcgccc gcgtctatca tcttgaccgc ttcgtcaaag 8760 gtgcaactgc cgctcttcaaacgccgcata gcggtcacgt cccgctctat gcacgacata 8820 ccgtttacgt acgattctgataggtattcc tgaactatac ggtaatggtg atacgactcg 8880 ccatacacgt cgtgcacctcattgtattta gcataataat tgtaaattat taactttgca 8940 gcgagagaca tgttgtcagtaaagcggtgc taggctcaat aatactgatg tacaggcacg 9000 cgtgctattt atatataatttcgcaaggag gggagctgtt atcggttgct attattaaag 9060 aatggccgtc tgtttttatcacaagcttgg cagcctcaac catgaagcgt cgtcattgta 9120 aattaaattc tctgcctcaagaattatttg acaagattgt cgagtattta tctttatctg 9180 attactgcaa tttggtgcttgtctgtaaaa gaccttctag taaatataac gtgatatttg 9240 atagtactaa tcaccaacatttgaaaggcg tgtacaaaaa gacagacgtg caaataacaa 9300 gctacaacga atacatcaactgtatttgca acgaactgag acaagacgaa ttctatgcca 9360 aatcatcatg gattgcgagtatttgcggtc accagagagc gacaattttt agtgtaacaa 9420 ataaacaagt agaaatgaaatatcatttgt ataatatagc aattgtggaa agtgaagatt 9480 gcaacggatt ttacccatttgagccaacgc gcgattgttt aatatgcaaa caaaaaaacc 9540 aatgtcctcg taattcatttattgtttcgt tgtgtaaata tttagaaaaa caaaatgtac 9600 aatcaaactt tatatattatttatacgaaa taaatacata ataataacta ttatacatgt 9660 ttttatttta caatacttcctgtataacct ctctaactac attaggagta caatccacgt 9720 caattacacg tttagctatttttctaattt tgtaatgttt atcgtagagt ttttcgttaa 9780 tacattgaat agccaacaagggatttgggt gcacaccgtc atagagtact tccatgtcgt 9840 cttcaaagcg catttttcgcttgcgaaaat gccgctcttg gcccaaaaca aaagcgagtt 9900 gatgcggtc gtcgatgcgttccgaaaata cggccaaatg ctggtgtttg gtgatgtcgc 9960 cggaaacgt caccgtgccatttttgcttt ccgccacgac 10000 3 10000 DNA Artificial Sequence Polyhedrinlocus of vBacHTS_HisGst 3 gaattctacc cgtaaagcga gtttagtttt gaaaaacaaatgacatcatt tgtataatga 60 catcatcccc tgattgtgtt ttacaagtag aattctatccgtaaagcgag ttcagttttg 120 aaaacaaatg agtcatacct aaacacgtta ataatcttctgatatcagct tatgactcaa 180 gttatgagcc gtgtgcaaaa catgagataa gtttatgacatcatccactg atcgtgcgtt 240 acaagtagaa ttctactcgt aaagccagtt cggttatgagccgtgtgcaa aacatgacat 300 cagcttatga ctcatacttg attgtgtttt acgcgtagaattctactcgt aaagcgagtt 360 cggttatgag ccgtgtgcaa aacatgacat cagcttatgagtcataatta atcgtgcgtt 420 acaagtagaa ttctactcgt aaagcgagtt gaaggatcatatttagttgc gtttatgaga 480 taagattgaa agcacgtgta aaatgtttcc cgcgcgttggcacaactatt tacaatgcgg 540 ccaagttata aaagattcta atctgatatg ttttaaaacacctttgcggc ccgagttgtt 600 tgcgtacgtg actagcgaag aagatgtgtg gaccgcagaacagatagtaa aacaaaaccc 660 tagtattgga gcaataatcg atttaaccaa cacgtctaaatattatgatg gtgtgcattt 720 tttgcgggcg ggcctgttat acaaaaaaat tcaagtacctggccagactt tgccgcctga 780 aagcatagtt caagaattta ttgacacggt aaaagaatttacagaaaagt gtcccggcat 840 gttggtgggc gtgcactgca cacacggtat taatcgcaccggttacatgg tgtgcagata 900 tttaatgcac accctgggta ttgcgccgca ggaagccatagatagattcg aaaaagccag 960 aggtcacaaa attgaaagac aaaattacgt tcaagatttattaatttaat taatattatt 1020 tgcattcttt aacaaatact ttatcctatt ttcaaattgttgcgcttctt ccagcgaacc 1080 aaaactatgc ttcgcttgct ccgtttagct tgtagccgatcagtggcgtt gttccaatcg 1140 acggtaggat taggccggat attctccacc acaatgttggcaacgttgat gttacgttta 1200 tgcttttggt tttccacgta cgtcttttgg ccggtaatagccgtaaacgt agtgccgtcg 1260 cgcgtcacgc acaacaccgg atgtttgcgc ttgtccgcggggtattgaac cgcgcgatcc 1320 gacaaatcca ccactttggc aactaaatcg gtgacctgcgcgtctttttt ctgcattatt 1380 tcgtctttct tttgcatggt ttcctggaag ccggtgtacatgcggtttag atcagtcatg 1440 acgcgcgtga cctgcaaatc tttggcctcg atctgcttgtccttgatggc aacgatgcgt 1500 tcaataaact cttgtttttt aacaagttcc tcggttttttgcgccaccac cgcttgcagc 1560 gcgtttgtgt gctcggtgaa tgtcgcaatc agcttagtcaccaactgttt gctctcctcc 1620 tcccgttgtt tgatcgcggg atcgtacttg ccggtgcagagcacttgagg aattacttct 1680 tctaaaagcc attcttgtaa ttctatggcg taaggcaatttggacttcat aatcagctga 1740 atcacgccgg atttagtaat gagcactgta tgcggctgcaaatacagcgg gtcgcccctt 1800 ttcacgacgc tgttagaggt agggccccca ttttggatggtctgctcaaa taacgatttg 1860 tatttattgt ctacatgaac acgtatagct ttatcacaaactgtatattt taaactgtta 1920 gcgacgtcct tggccacgaa ccggacctgt tggtcgcgctctagcacgta ccgcaggttg 1980 aacgtatctt ctccaaattt aaattctcca attttaacgcgagccatttt gatacacgtg 2040 tgtcgatttt gcaacaacta ttgtttttta acgcaaactaaacttattgt ggtaagcaat 2100 aattaaatat gggggaacat gcgccgctac aacactcgtcgttatgaacg cagacggcgc 2160 cggtctcggc gcaagcggct aaaacgtgtt gcgcgttcaacgcggcaaac atcgcaaaag 2220 ccaatagtac agttttgatt tgcatattaa cggcgattttttaaattatc ttatttaata 2280 aatagttatg acgcctacaa ctccccgccc gcgttgactcgctgcacctc gagcagttcg 2340 ttgacgcctt cctccgtgtg gccgaacacg tcgagcgggtggtcgatgac cagcggcgtg 2400 ccgcacgcga cgcacaagta tctgtacacc gaatgatcgtcgggcgaagg cacgtcggcc 2460 tccaagtggc aatattggca aattcgaaaa tatatacagttgggttgttt gcgcatatct 2520 atcgtggcgt tgggcatgta cgtccgaacg ttgatttgcatgcaagccga aattaaatca 2580 ttgcgattag tgcgattaaa acgttgtaca tcctcgcttttaatcatgcc gtcgattaaa 2640 tcgcgcaatc gagtcaagtg atcaaagtgt ggaataatgttttctttgta ttcccgagtc 2700 aagcgcagcg cgtattttaa caaactagcc atcttgtaagttagtttcat ttaatgcaac 2760 tttatccaat aatatattat gtatcgcacg tcaagaattaacaatgcgcc cgttgtcgca 2820 tctcaacacg actatgatag agatcaaata aagcgcgaattaaatagctt gcgacgcaac 2880 gtgcacgatc tgtgcacgcg ttccggcacg agctttgattgtaataagtt tttacgaagc 2940 gatgacatga cccccgtagt gacaacgatc acgcccaaaagaactgccga ctacaaaatt 3000 accgagtatg tcggtgacgt taaaactatt aagccatccaatcgaccgtt agtcgaatca 3060 ggaccgctgg tgcgagaagc cgcgaagtat ggcgaatgcatcgtataacg tgtggagtcc 3120 gctcattaga gcgtcatgtt tagacaagaa agctacatatttaattgatc ccgatgattt 3180 tattgataaa ttgaccctaa ctccatacac ggtattctacaatggcgggg ttttggtcaa 3240 aatttccgga ctgcgattgt acatgctgtt aacggctccgcccactatta atgaaattaa 3300 aaattccaat tttaaaaaac gcagcaagag aaacatttgtatgaaagaat gcgtagaagg 3360 aaagaaaaat gtcgtcgaca tgctgaacaa caagattaatatgcctccgt gtataaaaaa 3420 aatattgaac gatttgaaag aaaacaatgt accgcgcggcggtatgtaca ggaagaggtt 3480 tatactaaac tgttacattg caaacgtggt ttcgtgtgccaagtgtgaaa accgatgttt 3540 aatcaaggct ctgacgcatt tctacaacca cgactccaagtgtgtgggtg aagtcatgca 3600 tcttttaatc aaatcccaag atgtgtataa accaccaaactgccaaaaaa tgaaaactgt 3660 cgacaagctc tgtccgtttg ctggcaactg caagggtctcaatcctattt gtaattattg 3720 aataataaaa caattataaa tgctaaattt gttttttattaacgatacaa accaaacgca 3780 acaagaacat ttgtagtatt atctataatt gaaaacgcgtagttataatc gctgaggtaa 3840 tatttaaaat cattttcaaa tgattcacag ttaatttgcgacaatataat tttattttca 3900 cataaactag acgccttgtc gtcttcttct tcgtattccttctctttttc atttttctcc 3960 tcataaaaat taacatagtt attatcgtat ccatatatgtatctatcgta tagagtaaat 4020 tttttgttgt cataaatata tatgtctttt ttaatggggtgtatagtacc gctgcgcata 4080 gtttttctgt aatttacaac agtgctattt tctggtagttcttcggagtg tgttgcttta 4140 attattaaat ttatataatc aatgaatttg ggatcgtcggttttgtacaa tatgttgccg 4200 gcatagtacg cagcttcttc tagttcaatt acaccattttttagcagcac cggattaaca 4260 taactttcca aaatgttgta cgaaccgtta aacaaaaacagttcacctcc cttttctata 4320 ctattgtctg cgagcagttg tttgttgtta aaaataacagccattgtaat gagacgcaca 4380 aactaatatc acaaactgga aatgtctatc aatatatagttgctgatatc atggagataa 4440 ttaaaatgat aaccatctcg caaataaata agtattttactgttttcgta acagttttgt 4500 aataaaaaaa cctataaata cggatctatg catcatcaccatcatcacgg atctatgtcc 4560 cctatactag gttattggaa aattaagggc cttgtgcaacccactcgact tcttttggaa 4620 tatcttgaag aaaaatatga agagcatttg tatgagcgcgatgaaggtga taaatggcga 4680 aacaaaaagt ttgaattggg tttggagttt cccaatcttccttattatat tgatggtgat 4740 gttaaattaa cacagtctat ggccatcata cgttatatagctgacaagca caacatgttg 4800 ggtggttgtc caaaagagcg tgcagagatt tcaatgcttgaaggagcggt tttggatatt 4860 agatacggtg tttcgagaat tgcatatagt aaagactttgaaactctcaa agttgatttt 4920 cttagcaagc tacctgaaat gctgaaaatg ttcgaagatcgtttatgtca taaaacatat 4980 ttaaatggtg atcatgtaac ccatcctgac ttcatgttgtatgacgctct tgatgttgtt 5040 ttatacatgg acccaatgtg cctggatgcg ttcccaaaattagtttgttt taaaaaacgt 5100 attgaagcta tcccacaaat tgataagtac ttgaaatccagcaagtatat agcatggcct 5160 ttgcagggct ggcaagccac gtttggtggt ggcgaccatcctccaaaatc ggatctggtt 5220 ccgcgtggat cccggggtac cacaagtttg tacaaaaaagctgaacgaga aacgtaaaat 5280 gatataaata tcaatatatt aaattagatt ttgcataaaaaacagactac ataatactgt 5340 aaaacacaac atatccagtc actatggcgg ccgcattaggcaccccttag gaattcgctt 5400 actaaaagcc agataacagt atgcgtattt gcgcgctgatttttgcggta taagaatata 5460 tactgatatg tatacccgaa gtatgtcaaa aagaggtgtgcttctagaat gcagtttaag 5520 gtttacacct ataaaagaga gagccgttat cgtctgtttgtggatgtaca gagtgatatt 5580 attgacacgc ccgggcgacg gatggtgatc cccctggccagtgcacgtct gctgtcagat 5640 aaagtctccc gtgaacttta cccggtggtg catatcggggatgaaagctg gcgcatgatg 5700 accaccgata tggccagtgt gccggtctcc gttatcggggaagaagtggc tgatctcagc 5760 caccgcgaaa atgacatcaa aaacgccatt aacctgatgttctggggaat atagaattcc 5820 tcaggtcgac catagtgact ggatatgttg tgttttacagtattatgtag tctgtttttt 5880 atgcaaaatc taatttaata tattgatatt tatatcattttacgtttctc gttcagcttt 5940 cttgtacaaa gtggtctcga gttaattaat tgatccgggttattagtaca tttattaagc 6000 gctagattct gtgcgttgtt gatttacaga caattgttgtacgtatttta ataattcatt 6060 aaatttataa tctttagggt ggtatgttag agcgaaaatcaaatgatttt cagcgtcttt 6120 atatctgaat ttaaatatta aatcctcaat agatttgtaaaataggtttc gattagtttc 6180 aaacaagggt tgtttttccg aaccgatggc tggactatctaatggatttt cgctcaacgc 6240 cacaaaactt gccaaatctt gtagcagcaa tctagctttgtcgatattcg tttgtgtttt 6300 gttttgtaat aaaggttcga cgtcgttcaa aatattatgcgcttttgtat ttctttcatc 6360 actgtcgtta gtgtacaatt gactcgacgt aaacacgttaaataaagctt ggacatattt 6420 aacatcgggc gtgttagctt tattaggccg attatcgtcgtcgtcccaac cctcgtcgtt 6480 agaagttgct tccgaagacg attttgccat agccacacgacgcctattaa ttgtgtcggc 6540 taacacgtcc gcgatcaaat ttgtagttga gctttttggaattatttctg attgcgggcg 6600 tttttgggcg ggtttcaatc taactgtgcc cgattttaattcagacaaca cgttagaaag 6660 cgatggtgca ggcggtggta acatttcaga cggcaaatctactaatggcg gcggtggtgg 6720 agctgatgat aaatctacca tcggtggagg cgcaggcggggctggcggcg gaggcggagg 6780 cggaggtggt ggcggtgatg cagacggcgg tttaggctcaaatgtctctt taggcaacac 6840 agtcggcacc tcaactattg tactggtttc gggcgccgtttttggtttga ccggtctgag 6900 acgagtgcga tttttttcgt ttctaatagc ttccaacaattgttgtctgt cgtctaaagg 6960 tgcagcgggt tgaggttccg tcggcattgg tggagcgggcggcaattcag acatcgatgg 7020 tggtggtggt ggtggaggcg ctggaatgtt aggcacgggagaaggtggtg gcggcggtgc 7080 cgccggtata atttgttctg gtttagtttg ttcgcgcacgattgtgggca ccggcgcagg 7140 cgccgctggc tgcacaacgg aaggtcgtct gcttcgaggcagcgcttggg gtggtggcaa 7200 ttcaatatta taattggaat acaaatcgta aaaatctgctataagcattg taatttcgct 7260 atcgtttacc gtgccgatat ttaacaaccg ctcaatgtaagcaattgtat tgtaaagaga 7320 ttgtctcaag ctcggatccc gcacgccgat aacaagccttttcattttta ctacagcatt 7380 gtagtggcga gacacttcgc tgtcgtcgac gtacatgtatgctttgttgt caaaaacgtc 7440 gttggcaagc tttaaaatat ttaaaagaac atctctgttcagcaccactg tgttgtcgta 7500 aatgttgttt ttgataattt gcgcttccgc agtatcgacacgttcaaaaa attgatgcgc 7560 atcaattttg ttgttcctat tattgaataa ataagattgtacagattcat atctacgatt 7620 cgtcatggcc accacaaatg ctacgctgca aacgctggtacaattttacg aaaactgcaa 7680 aaacgtcaaa actcggtata aaataatcaa cgggcgctttggcaaaatat ctattttatc 7740 gcacaagccc actagcaaat tgtatttgca gaaaacaatttcggcgcaca attttaacgc 7800 tgacgaaata aaagttcacc agttaatgag cgaccacccaaattttataa aaatctattt 7860 taatcacggt tccatcaaca accaagtgat cgtgatggactacattgact gtcccgattt 7920 atttgaaaca ctacaaatta aaggcgagct ttcgtaccaacttgttagca atattattag 7980 acagctgtgt gaagcgctca acgatttgca caagcacaatttcatacaca acgacataaa 8040 actcgaaaat gtcttatatt tcgaagcact tgatcgcgtgtatgtttgcg attacggatt 8100 gtgcaaacac gaaaactcac ttagcgtgca cgacggcacgttggagtatt ttagtccgga 8160 aaaaattcga cacacaacta tgcacgtttc gtttgactggtacgccgtcg gcgtgttaac 8220 atacaagttg ctaaccggcg gccgacaccc atttgaaaaaagcgaagacg aaatgttgga 8280 cttgaatagc atgaagcgtc gtcagcaata caatgacattggcgttttaa aacacgttcg 8340 taacgttaac gctcgtgact ttgtgtactg cctaacaagatacaacatag attgtagact 8400 cacaaattac aaacaaatta taaaacatga gtttttgtcgtaaaaatgcc acttgtttta 8460 cgagtagaat tctacgtgta acacacgatc taaaagatgatgtcattttt tatcaatgac 8520 tcatttgttt taaaacagac ttgttttacg agtagaattctacgtgtaaa gcatgatcgt 8580 gagtggtgtt aataaaatca taaaaattat tgtaaatgtttattatttaa aaacgattca 8640 aatatataat aaaaacaatc tacatctatt tcttcacaatccataacaca caacaggtcc 8700 atcaatgagt ttttgtcttt atccgacata ctatgtgcatgtaacaaatc aaatacatct 8760 tttaaatttt tatacacatc tttacattgt ctaccaaaatctttaataac cctataacaa 8820 ggaaaagact tttcttcttg cgtggttttg ccgcgcagatattgaaataa aatgtgcatg 8880 cacgacaact tgtgtttact aaaatgctcc ttgcctataccgcaaaaccg gccatacatt 8940 tcggcgatta cacgcggaca attgtacgat tcgtctacgtgtaaacgatc atcataatca 9000 ctcttgcgca aacgaataaa ttttttcacc gcttccgacaaacgaggcac caattcggcg 9060 ggcacgcttc gatacattat tctgtgcaca taagttaccacacaaaattt attgtaccac 9120 catccgacaa cgtcgttatt agggttgaac acgttggcgatgcgcagcag tttcccgttt 9180 ctcatgaaat attcaaagcg gcccaaaata atttgcaagcaatccaacat gtcttgagaa 9240 atttctcgtt caaaattgtt caaagagaat atctgccatccgttttgaac gcgcacgctg 9300 acgggaacca ccgcatcgat ttgctccaac acttcacggacgttatcgtc gatgcccatc 9360 gtttcgctgg tgctgaacca atgggaaagg ctcttgatggaatcgcccgc gtctatcatc 9420 ttgaccgctt cgtcaaaggt gcaactgccg ctcttcaaacgccgcatagc ggtcacgtcc 9480 cgctctatgc acgacatacc gtttacgtac gattctgataggtattcctg aactatacgg 9540 taatggtgat acgactcgcc atacacgtcg tgcacctcattgtatttagc ataataattg 9600 taaattatta actttgcagc gagagacatg ttgtcagtaaagcggtgcta ggctcaataa 9660 tactgatgta caggcacgcg tgctatttat atataatttcgcaaggaggg gagctgttat 9720 cggttgctat tattaaagaa tggccgtctg tttttatcacaagcttggca gcctcaacca 9780 tgaagcgtcg tcattgtaaa ttaaattctc tgcctcaagaattatttgac aagattgtcg 9840 agtatttatc tttatctgat tactgcaatt tggtgcttgtctgtaaaaga ccttctagta 9900 aatataacgt gatatttgat agtactaatc accaacatttgaaaggcgtg tacaaaaaga 9960 cagacgtgca aataacaagc tacaacgaat acatcaactg10000 4 10000 DNA Artificial Sequence Polyhedrin locus of vBacHTS_GST 4gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa tgacatcatt tgtataatga 60catcatcccc tgattgtgtt ttacaagtag aattctatcc gtaaagcgag ttcagttttg 120aaaacaaatg agtcatacct aaacacgtta ataatcttct gatatcagct tatgactcaa 180gttatgagcc gtgtgcaaaa catgagataa gtttatgaca tcatccactg atcgtgcgtt 240acaagtagaa ttctactcgt aaagccagtt cggttatgag ccgtgtgcaa aacatgacat 300cagcttatga ctcatacttg attgtgtttt acgcgtagaa ttctactcgt aaagcgagtt 360cggttatgag ccgtgtgcaa aacatgacat cagcttatga gtcataatta atcgtgcgtt 420acaagtagaa ttctactcgt aaagcgagtt gaaggatcat atttagttgc gtttatgaga 480taagattgaa agcacgtgta aaatgtttcc cgcgcgttgg cacaactatt tacaatgcgg 540ccaagttata aaagattcta atctgatatg ttttaaaaca cctttgcggc ccgagttgtt 600tgcgtacgtg actagcgaag aagatgtgtg gaccgcagaa cagatagtaa aacaaaaccc 660tagtattgga gcaataatcg atttaaccaa cacgtctaaa tattatgatg gtgtgcattt 720tttgcgggcg ggcctgttat acaaaaaaat tcaagtacct ggccagactt tgccgcctga 780aagcatagtt caagaattta ttgacacggt aaaagaattt acagaaaagt gtcccggcat 840gttggtgggc gtgcactgca cacacggtat taatcgcacc ggttacatgg tgtgcagata 900tttaatgcac accctgggta ttgcgccgca ggaagccata gatagattcg aaaaagccag 960aggtcacaaa attgaaagac aaaattacgt tcaagattta ttaatttaat taatattatt 1020tgcattcttt aacaaatact ttatcctatt ttcaaattgt tgcgcttctt ccagcgaacc 1080aaaactatgc ttcgcttgct ccgtttagct tgtagccgat cagtggcgtt gttccaatcg 1140acggtaggat taggccggat attctccacc acaatgttgg caacgttgat gttacgttta 1200tgcttttggt tttccacgta cgtcttttgg ccggtaatag ccgtaaacgt agtgccgtcg 1260cgcgtcacgc acaacaccgg atgtttgcgc ttgtccgcgg ggtattgaac cgcgcgatcc 1320gacaaatcca ccactttggc aactaaatcg gtgacctgcg cgtctttttt ctgcattatt 1380tcgtctttct tttgcatggt ttcctggaag ccggtgtaca tgcggtttag atcagtcatg 1440acgcgcgtga cctgcaaatc tttggcctcg atctgcttgt ccttgatggc aacgatgcgt 1500tcaataaact cttgtttttt aacaagttcc tcggtttttt gcgccaccac cgcttgcagc 1560gcgtttgtgt gctcggtgaa tgtcgcaatc agcttagtca ccaactgttt gctctcctcc 1620tcccgttgtt tgatcgcggg atcgtacttg ccggtgcaga gcacttgagg aattacttct 1680tctaaaagcc attcttgtaa ttctatggcg taaggcaatt tggacttcat aatcagctga 1740atcacgccgg atttagtaat gagcactgta tgcggctgca aatacagcgg gtcgcccctt 1800ttcacgacgc tgttagaggt agggccccca ttttggatgg tctgctcaaa taacgatttg 1860tatttattgt ctacatgaac acgtatagct ttatcacaaa ctgtatattt taaactgtta 1920gcgacgtcct tggccacgaa ccggacctgt tggtcgcgct ctagcacgta ccgcaggttg 1980aacgtatctt ctccaaattt aaattctcca attttaacgc gagccatttt gatacacgtg 2040tgtcgatttt gcaacaacta ttgtttttta acgcaaacta aacttattgt ggtaagcaat 2100aattaaatat gggggaacat gcgccgctac aacactcgtc gttatgaacg cagacggcgc 2160cggtctcggc gcaagcggct aaaacgtgtt gcgcgttcaa cgcggcaaac atcgcaaaag 2220ccaatagtac agttttgatt tgcatattaa cggcgatttt ttaaattatc ttatttaata 2280aatagttatg acgcctacaa ctccccgccc gcgttgactc gctgcacctc gagcagttcg 2340ttgacgcctt cctccgtgtg gccgaacacg tcgagcgggt ggtcgatgac cagcggcgtg 2400ccgcacgcga cgcacaagta tctgtacacc gaatgatcgt cgggcgaagg cacgtcggcc 2460tccaagtggc aatattggca aattcgaaaa tatatacagt tgggttgttt gcgcatatct 2520atcgtggcgt tgggcatgta cgtccgaacg ttgatttgca tgcaagccga aattaaatca 2580ttgcgattag tgcgattaaa acgttgtaca tcctcgcttt taatcatgcc gtcgattaaa 2640tcgcgcaatc gagtcaagtg atcaaagtgt ggaataatgt tttctttgta ttcccgagtc 2700aagcgcagcg cgtattttaa caaactagcc atcttgtaag ttagtttcat ttaatgcaac 2760tttatccaat aatatattat gtatcgcacg tcaagaatta acaatgcgcc cgttgtcgca 2820tctcaacacg actatgatag agatcaaata aagcgcgaat taaatagctt gcgacgcaac 2880gtgcacgatc tgtgcacgcg ttccggcacg agctttgatt gtaataagtt tttacgaagc 2940gatgacatga cccccgtagt gacaacgatc acgcccaaaa gaactgccga ctacaaaatt 3000accgagtatg tcggtgacgt taaaactatt aagccatcca atcgaccgtt agtcgaatca 3060ggaccgctgg tgcgagaagc cgcgaagtat ggcgaatgca tcgtataacg tgtggagtcc 3120gctcattaga gcgtcatgtt tagacaagaa agctacatat ttaattgatc ccgatgattt 3180tattgataaa ttgaccctaa ctccatacac ggtattctac aatggcgggg ttttggtcaa 3240aatttccgga ctgcgattgt acatgctgtt aacggctccg cccactatta atgaaattaa 3300aaattccaat tttaaaaaac gcagcaagag aaacatttgt atgaaagaat gcgtagaagg 3360aaagaaaaat gtcgtcgaca tgctgaacaa caagattaat atgcctccgt gtataaaaaa 3420aatattgaac gatttgaaag aaaacaatgt accgcgcggc ggtatgtaca ggaagaggtt 3480tatactaaac tgttacattg caaacgtggt ttcgtgtgcc aagtgtgaaa accgatgttt 3540aatcaaggct ctgacgcatt tctacaacca cgactccaag tgtgtgggtg aagtcatgca 3600tcttttaatc aaatcccaag atgtgtataa accaccaaac tgccaaaaaa tgaaaactgt 3660cgacaagctc tgtccgtttg ctggcaactg caagggtctc aatcctattt gtaattattg 3720aataataaaa caattataaa tgctaaattt gttttttatt aacgatacaa accaaacgca 3780acaagaacat ttgtagtatt atctataatt gaaaacgcgt agttataatc gctgaggtaa 3840tatttaaaat cattttcaaa tgattcacag ttaatttgcg acaatataat tttattttca 3900cataaactag acgccttgtc gtcttcttct tcgtattcct tctctttttc atttttctcc 3960tcataaaaat taacatagtt attatcgtat ccatatatgt atctatcgta tagagtaaat 4020tttttgttgt cataaatata tatgtctttt ttaatggggt gtatagtacc gctgcgcata 4080gtttttctgt aatttacaac agtgctattt tctggtagtt cttcggagtg tgttgcttta 4140attattaaat ttatataatc aatgaatttg ggatcgtcgg ttttgtacaa tatgttgccg 4200gcatagtacg cagcttcttc tagttcaatt acaccatttt ttagcagcac cggattaaca 4260taactttcca aaatgttgta cgaaccgtta aacaaaaaca gttcacctcc cttttctata 4320ctattgtctg cgagcagttg tttgttgtta aaaataacag ccattgtaat gagacgcaca 4380aactaatatc acaaactgga aatgtctatc aatatatagt tgctgatatc atggagataa 4440ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt 4500aataaaaaaa cctataaata cggatct atg tcc cct ata cta ggt tat tgg 4551 MetSer Pro Ile Leu Gly Tyr Trp 1 5 aaa att aag ggc ctt gtg caa ccc act cgactt ctt ttg gaa tat ctt 4599 Lys Ile Lys Gly Leu Val Gln Pro Thr Arg LeuLeu Leu Glu Tyr Leu 10 15 20 gaa gaa aaa tat gaa gag cat ttg tat gag cgcgat gaa ggt gat aaa 4647 Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg AspGlu Gly Asp Lys 25 30 35 40 tgg cga aac aaa aag ttt gaa ttg ggt ttg gagttt ccc aat ctt cct 4695 Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu PhePro Asn Leu Pro 45 50 55 tat tat att gat ggt gat gtt aaa tta aca cag tctatg gcc atc ata 4743 Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser MetAla Ile Ile 60 65 70 cgt tat ata gct gac aag cac aac atg ttg ggt ggt tgtcca aaa gag 4791 Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys ProLys Glu 75 80 85 cgt gca gag att tca atg ctt gaa gga gcg gtt ttg gat attaga tac 4839 Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile ArgTyr 90 95 100 ggt gtt tcg aga att gca tat agt aaa gac ttt gaa act ctcaaa gtt 4887 Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu LysVal 105 110 115 120 gat ttt ctt agc aag cta cct gaa atg ctg aaa atg ttcgaa gat cgt 4935 Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe GluAsp Arg 125 130 135 tta tgt cat aaa aca tat tta aat ggt gat cat gta acccat cct gac 4983 Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr HisPro Asp 140 145 150 ttc atg ttg tat gac gct ctt gat gtt gtt tta tac atggac cca atg 5031 Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met AspPro Met 155 160 165 tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa aaacgt att gaa 5079 Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys ArgIle Glu 170 175 180 gct atc cca caa att gat aag tac ttg aaa tcc agc aagtat ata gca 5127 Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys TyrIle Ala 185 190 195 200 tgg cct ttg cag ggc tgg caa gcc acg ttt ggt ggtggc gac cat cct 5175 Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly GlyAsp His Pro 205 210 215 cca aaa tcggatctg gttccgcgtg gatcccggggtaccacaagt ttgtacaaaa 5230 Pro Lys aagctgaacg agaaacgtaa aatgatataaatatcaatat attaaattag attttgcata 5290 aaaaacagac tacataatac tgtaaaacacaacatatcca gtcactatgg cggccgcatt 5350 aggcacccct taggaattcg cttactaaaagccagataac agtatgcgta tttgcgcgct 5410 gatttttgcg gtataagaat atatactgatatgtataccc gaagtatgtc aaaaagaggt 5470 gtgcttctag aatgcagttt aaggtttacacctataaaag agagagccgt tatcgtctgt 5530 ttgtggatgt acagagtgat attattgacacgcccgggcg acggatggtg atccccctgg 5590 ccagtgcacg tctgctgtca gataaagtctcccgtgaact ttacccggtg gtgcatatcg 5650 gggatgaaag ctggcgcatg atgaccaccgatatggccag tgtgccggtc tccgttatcg 5710 gggaagaagt ggctgatctc agccaccgcgaaaatgacat caaaaacgcc attaacctga 5770 tgttctgggg aatatagaat tcctcaggtcgaccatagtg actggatatg ttgtgtttta 5830 cagtattatg tagtctgttt tttatgcaaaatctaattta atatattgat atttatatca 5890 ttttacgttt ctcgttcagc tttcttgtacaaagtggtct cgagttaatt aattgatccg 5950 ggttattagt acatttatta agcgctagattctgtgcgtt gttgatttac agacaattgt 6010 tgtacgtatt ttaataattc attaaatttataatctttag ggtggtatgt tagagcgaaa 6070 atcaaatgat tttcagcgtc tttatatctgaatttaaata ttaaatcctc aatagatttg 6130 taaaataggt ttcgattagt ttcaaacaagggttgttttt ccgaaccgat ggctggacta 6190 tctaatggat tttcgctcaa cgccacaaaacttgccaaat cttgtagcag caatctagct 6250 ttgtcgatat tcgtttgtgt tttgttttgtaataaaggtt cgacgtcgtt caaaatatta 6310 tgcgcttttg tatttctttc atcactgtcgttagtgtaca attgactcga cgtaaacacg 6370 ttaaataaag cttggacata tttaacatcgggcgtgttag ctttattagg ccgattatcg 6430 tcgtcgtccc aaccctcgtc gttagaagttgcttccgaag acgattttgc catagccaca 6490 cgacgcctat taattgtgtc ggctaacacgtccgcgatca aatttgtagt tgagcttttt 6550 ggaattattt ctgattgcgg gcgtttttgggcgggtttca atctaactgt gcccgatttt 6610 aattcagaca acacgttaga aagcgatggtgcaggcggtg gtaacatttc agacggcaaa 6670 tctactaatg gcggcggtgg tggagctgatgataaatcta ccatcggtgg aggcgcaggc 6730 ggggctggcg gcggaggcgg aggcggaggtggtggcggtg atgcagacgg cggtttaggc 6790 tcaaatgtct ctttaggcaa cacagtcggcacctcaacta ttgtactggt ttcgggcgcc 6850 gtttttggtt tgaccggtct gagacgagtgcgattttttt cgtttctaat agcttccaac 6910 aattgttgtc tgtcgtctaa aggtgcagcgggttgaggtt ccgtcggcat tggtggagcg 6970 ggcggcaatt cagacatcga tggtggtggtggtggtggag gcgctggaat gttaggcacg 7030 ggagaaggtg gtggcggcgg tgccgccggtataatttgtt ctggtttagt ttgttcgcgc 7090 acgattgtgg gcaccggcgc aggcgccgctggctgcacaa cggaaggtcg tctgcttcga 7150 ggcagcgctt ggggtggtgg caattcaatattataattgg aatacaaatc gtaaaaatct 7210 gctataagca ttgtaatttc gctatcgtttaccgtgccga tatttaacaa ccgctcaatg 7270 taagcaattg tattgtaaag agattgtctcaagctcggat cccgcacgcc gataacaagc 7330 cttttcattt ttactacagc attgtagtggcgagacactt cgctgtcgtc gacgtacatg 7390 tatgctttgt tgtcaaaaac gtcgttggcaagctttaaaa tatttaaaag aacatctctg 7450 ttcagcacca ctgtgttgtc gtaaatgttgtttttgataa tttgcgcttc cgcagtatcg 7510 acacgttcaa aaaattgatg cgcatcaattttgttgttcc tattattgaa taaataagat 7570 tgtacagatt catatctacg attcgtcatggccaccacaa atgctacgct gcaaacgctg 7630 gtacaatttt acgaaaactg caaaaacgtcaaaactcggt ataaaataat caacgggcgc 7690 tttggcaaaa tatctatttt atcgcacaagcccactagca aattgtattt gcagaaaaca 7750 atttcggcgc acaattttaa cgctgacgaaataaaagttc accagttaat gagcgaccac 7810 ccaaatttta taaaaatcta ttttaatcacggttccatca acaaccaagt gatcgtgatg 7870 gactacattg actgtcccga tttatttgaaacactacaaa ttaaaggcga gctttcgtac 7930 caacttgtta gcaatattat tagacagctgtgtgaagcgc tcaacgattt gcacaagcac 7990 aatttcatac acaacgacat aaaactcgaaaatgtcttat atttcgaagc acttgatcgc 8050 gtgtatgttt gcgattacgg attgtgcaaacacgaaaact cacttagcgt gcacgacggc 8110 acgttggagt attttagtcc ggaaaaaattcgacacacaa ctatgcacgt ttcgtttgac 8170 tggtacgccg tcggcgtgtt aacatacaagttgctaaccg gcggccgaca cccatttgaa 8230 aaaagcgaag acgaaatgtt ggacttgaatagcatgaagc gtcgtcagca atacaatgac 8290 attggcgttt taaaacacgt tcgtaacgttaacgctcgtg actttgtgta ctgcctaaca 8350 agatacaaca tagattgtag actcacaaattacaaacaaa ttataaaaca tgagtttttg 8410 tcgtaaaaat gccacttgtt ttacgagtagaattctacgt gtaacacacg atctaaaaga 8470 tgatgtcatt ttttatcaat gactcatttgttttaaaaca gacttgtttt acgagtagaa 8530 ttctacgtgt aaagcatgat cgtgagtggtgttaataaaa tcataaaaat tattgtaaat 8590 gtttattatt taaaaacgat tcaaatatataataaaaaca atctacatct atttcttcac 8650 aatccataac acacaacagg tccatcaatgagtttttgtc tttatccgac atactatgtg 8710 catgtaacaa atcaaataca tcttttaaatttttatacac atctttacat tgtctaccaa 8770 aatctttaat aaccctataa caaggaaaagacttttcttc ttgcgtggtt ttgccgcgca 8830 gatattgaaa taaaatgtgc atgcacgacaacttgtgttt actaaaatgc tccttgccta 8890 taccgcaaaa ccggccatac atttcggcgattacacgcgg acaattgtac gattcgtcta 8950 cgtgtaaacg atcatcataa tcactcttgcgcaaacgaat aaattttttc accgcttccg 9010 acaaacgagg caccaattcg gcgggcacgcttcgatacat tattctgtgc acataagtta 9070 ccacacaaaa tttattgtac caccatccgacaacgtcgtt attagggttg aacacgttgg 9130 cgatgcgcag cagtttcccg tttctcatgaaatattcaaa gcggcccaaa ataatttgca 9190 agcaatccaa catgtcttga gaaatttctcgttcaaaatt gttcaaagag aatatctgcc 9250 atccgttttg aacgcgcacg ctgacgggaaccaccgcatc gatttgctcc aacacttcac 9310 ggacgttatc gtcgatgccc atcgtttcgctggtgctgaa ccaatgggaa aggctcttga 9370 tggaatcgcc cgcgtctatc atcttgaccgcttcgtcaaa ggtgcaactg ccgctcttca 9430 aacgccgcat agcggtcacg tcccgctctatgcacgacat accgtttacg tacgattctg 9490 ataggtattc ctgaactata cggtaatggtgatacgactc gccatacacg tcgtgcacct 9550 cattgtattt agcataataa ttgtaaattattaactttgc agcgagagac atgttgtcag 9610 taaagcggtg ctaggctcaa taatactgatgtacaggcac gcgtgctatt tatatataat 9670 ttcgcaagga ggggagctgt tatcggttgctattattaaa gaatggccgt ctgtttttat 9730 cacaagcttg gcagcctcaa ccatgaagcgtcgtcattgt aaattaaatt ctctgcctca 9790 agaattattt gacaagattg tcgagtatttatctttatct gattactgca atttggtgct 9850 tgtctgtaaa agaccttcta gtaaatataacgtgatattt gatagtacta atcaccaaca 9910 tttgaaaggc gtgtacaaaa agacagacgtgcaaataaca agctacaacg aatacatcaa 9970 ctgtatttgc aacgaactga gacaagacga10000 5 10000 DNA Artificial Sequence Polyhedrin locus of vBacHTS_GFPvirus 5 gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa tgacatcatttgtataatga 60 catcatcccc tgattgtgtt ttacaagtag aattctatcc gtaaagcgagttcagttttg 120 aaaacaaatg agtcatacct aaacacgtta ataatcttct gatatcagcttatgactcaa 180 gttatgagcc gtgtgcaaaa catgagataa gtttatgaca tcatccactgatcgtgcgtt 240 acaagtagaa ttctactcgt aaagccagtt cggttatgag ccgtgtgcaaaacatgacat 300 cagcttatga ctcatacttg attgtgtttt acgcgtagaa ttctactcgtaaagcgagtt 360 cggttatgag ccgtgtgcaa aacatgacat cagcttatga gtcataattaatcgtgcgtt 420 acaagtagaa ttctactcgt aaagcgagtt gaaggatcat atttagttgcgtttatgaga 480 taagattgaa agcacgtgta aaatgtttcc cgcgcgttgg cacaactatttacaatgcgg 540 ccaagttata aaagattcta atctgatatg ttttaaaaca cctttgcggcccgagttgtt 600 tgcgtacgtg actagcgaag aagatgtgtg gaccgcagaa cagatagtaaaacaaaaccc 660 tagtattgga gcaataatcg atttaaccaa cacgtctaaa tattatgatggtgtgcattt 720 tttgcgggcg ggcctgttat acaaaaaaat tcaagtacct ggccagactttgccgcctga 780 aagcatagtt caagaattta ttgacacggt aaaagaattt acagaaaagtgtcccggcat 840 gttggtgggc gtgcactgca cacacggtat taatcgcacc ggttacatggtgtgcagata 900 tttaatgcac accctgggta ttgcgccgca ggaagccata gatagattcgaaaaagccag 960 aggtcacaaa attgaaagac aaaattacgt tcaagattta ttaatttaattaatattatt 1020 tgcattcttt aacaaatact ttatcctatt ttcaaattgt tgcgcttcttccagcgaacc 1080 aaaactatgc ttcgcttgct ccgtttagct tgtagccgat cagtggcgttgttccaatcg 1140 acggtaggat taggccggat attctccacc acaatgttgg caacgttgatgttacgttta 1200 tgcttttggt tttccacgta cgtcttttgg ccggtaatag ccgtaaacgtagtgccgtcg 1260 cgcgtcacgc acaacaccgg atgtttgcgc ttgtccgcgg ggtattgaaccgcgcgatcc 1320 gacaaatcca ccactttggc aactaaatcg gtgacctgcg cgtcttttttctgcattatt 1380 tcgtctttct tttgcatggt ttcctggaag ccggtgtaca tgcggtttagatcagtcatg 1440 acgcgcgtga cctgcaaatc tttggcctcg atctgcttgt ccttgatggcaacgatgcgt 1500 tcaataaact cttgtttttt aacaagttcc tcggtttttt gcgccaccaccgcttgcagc 1560 gcgtttgtgt gctcggtgaa tgtcgcaatc agcttagtca ccaactgtttgctctcctcc 1620 tcccgttgtt tgatcgcggg atcgtacttg ccggtgcaga gcacttgaggaattacttct 1680 tctaaaagcc attcttgtaa ttctatggcg taaggcaatt tggacttcataatcagctga 1740 atcacgccgg atttagtaat gagcactgta tgcggctgca aatacagcgggtcgcccctt 1800 ttcacgacgc tgttagaggt agggccccca ttttggatgg tctgctcaaataacgatttg 1860 tatttattgt ctacatgaac acgtatagct ttatcacaaa ctgtatattttaaactgtta 1920 gcgacgtcct tggccacgaa ccggacctgt tggtcgcgct ctagcacgtaccgcaggttg 1980 aacgtatctt ctccaaattt aaattctcca attttaacgc gagccattttgatacacgtg 2040 tgtcgatttt gcaacaacta ttgtttttta acgcaaacta aacttattgtggtaagcaat 2100 aattaaatat gggggaacat gcgccgctac aacactcgtc gttatgaacgcagacggcgc 2160 cggtctcggc gcaagcggct aaaacgtgtt gcgcgttcaa cgcggcaaacatcgcaaaag 2220 ccaatagtac agttttgatt tgcatattaa cggcgatttt ttaaattatcttatttaata 2280 aatagttatg acgcctacaa ctccccgccc gcgttgactc gctgcacctcgagcagttcg 2340 ttgacgcctt cctccgtgtg gccgaacacg tcgagcgggt ggtcgatgaccagcggcgtg 2400 ccgcacgcga cgcacaagta tctgtacacc gaatgatcgt cgggcgaaggcacgtcggcc 2460 tccaagtggc aatattggca aattcgaaaa tatatacagt tgggttgtttgcgcatatct 2520 atcgtggcgt tgggcatgta cgtccgaacg ttgatttgca tgcaagccgaaattaaatca 2580 ttgcgattag tgcgattaaa acgttgtaca tcctcgcttt taatcatgccgtcgattaaa 2640 tcgcgcaatc gagtcaagtg atcaaagtgt ggaataatgt tttctttgtattcccgagtc 2700 aagcgcagcg cgtattttaa caaactagcc atcttgtaag ttagtttcatttaatgcaac 2760 tttatccaat aatatattat gtatcgcacg tcaagaatta acaatgcgcccgttgtcgca 2820 tctcaacacg actatgatag agatcaaata aagcgcgaat taaatagcttgcgacgcaac 2880 gtgcacgatc tgtgcacgcg ttccggcacg agctttgatt gtaataagtttttacgaagc 2940 gatgacatga cccccgtagt gacaacgatc acgcccaaaa gaactgccgactacaaaatt 3000 accgagtatg tcggtgacgt taaaactatt aagccatcca atcgaccgttagtcgaatca 3060 ggaccgctgg tgcgagaagc cgcgaagtat ggcgaatgca tcgtataacgtgtggagtcc 3120 gctcattaga gcgtcatgtt tagacaagaa agctacatat ttaattgatcccgatgattt 3180 tattgataaa ttgaccctaa ctccatacac ggtattctac aatggcggggttttggtcaa 3240 aatttccgga ctgcgattgt acatgctgtt aacggctccg cccactattaatgaaattaa 3300 aaattccaat tttaaaaaac gcagcaagag aaacatttgt atgaaagaatgcgtagaagg 3360 aaagaaaaat gtcgtcgaca tgctgaacaa caagattaat atgcctccgtgtataaaaaa 3420 aatattgaac gatttgaaag aaaacaatgt accgcgcggc ggtatgtacaggaagaggtt 3480 tatactaaac tgttacattg caaacgtggt ttcgtgtgcc aagtgtgaaaaccgatgttt 3540 aatcaaggct ctgacgcatt tctacaacca cgactccaag tgtgtgggtgaagtcatgca 3600 tcttttaatc aaatcccaag atgtgtataa accaccaaac tgccaaaaaatgaaaactgt 3660 cgacaagctc tgtccgtttg ctggcaactg caagggtctc aatcctatttgtaattattg 3720 aataataaaa caattataaa tgctaaattt gttttttatt aacgatacaaaccaaacgca 3780 acaagaacat ttgtagtatt atctataatt gaaaacgcgt agttataatcgctgaggtaa 3840 tatttaaaat cattttcaaa tgattcacag ttaatttgcg acaatataattttattttca 3900 cataaactag acgccttgtc gtcttcttct tcgtattcct tctctttttcatttttctcc 3960 tcataaaaat taacatagtt attatcgtat ccatatatgt atctatcgtatagagtaaat 4020 tttttgttgt cataaatata tatgtctttt ttaatggggt gtatagtaccgctgcgcata 4080 gtttttctgt aatttacaac agtgctattt tctggtagtt cttcggagtgtgttgcttta 4140 attattaaat ttatataatc aatgaatttg ggatcgtcgg ttttgtacaatatgttgccg 4200 gcatagtacg cagcttcttc tagttcaatt acaccatttt ttagcagcaccggattaaca 4260 taactttcca aaatgttgta cgaaccgtta aacaaaaaca gttcacctcccttttctata 4320 ctattgtctg cgagcagttg tttgttgtta aaaataacag ccattgtaatgagacgcaca 4380 aactaatatc acaaactgga aatgtctatc aatatatagt tgctgatatcatggagataa 4440 ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgtaacagttttgt 4500 aataaaaaaa cctataaata cggatccacc atg gtg agc aag ggc gaggag ctg 4554 Met Val Ser Lys Gly Glu Glu Leu 1 5 ttc acc ggg gtg gtg cccatc ctg gtc gag ctg gac ggc gac gta aac 4602 Phe Thr Gly Val Val Pro IleLeu Val Glu Leu Asp Gly Asp Val Asn 10 15 20 ggc cac aag ttc agc gtg tccggc gag ggc gag ggc gat gcc acc tac 4650 Gly His Lys Phe Ser Val Ser GlyGlu Gly Glu Gly Asp Ala Thr Tyr 25 30 35 40 ggc aag ctg acc ctg aag ttcatc tgc acc acc ggc aag ctg ccc gtg 4698 Gly Lys Leu Thr Leu Lys Phe IleCys Thr Thr Gly Lys Leu Pro Val 45 50 55 ccc tgg ccc acc ctc gtg acc accctg acc tac ggc gtg cag tgc ttc 4746 Pro Trp Pro Thr Leu Val Thr Thr LeuThr Tyr Gly Val Gln Cys Phe 60 65 70 agc cgc tac ccc gac cac atg aag cagcac gac ttc ttc aag tcc gcc 4794 Ser Arg Tyr Pro Asp His Met Lys Gln HisAsp Phe Phe Lys Ser Ala 75 80 85 atg ccc gaa ggc tac gtc cag gag cgc accatc ttc ttc aag gac gac 4842 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr IlePhe Phe Lys Asp Asp 90 95 100 ggc aac tac aag acc cgc gcc gag gtg aagttc gag ggc gac acc ctg 4890 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys PheGlu Gly Asp Thr Leu 105 110 115 120 gtg aac cgc atc gag ctg aag ggc atcgac ttc aag gag gac ggc aac 4938 Val Asn Arg Ile Glu Leu Lys Gly Ile AspPhe Lys Glu Asp Gly Asn 125 130 135 atc ctg ggg cac aag ctg gag tac aactac aac agc cac aac gtc tat 4986 Ile Leu Gly His Lys Leu Glu Tyr Asn TyrAsn Ser His Asn Val Tyr 140 145 150 atc atg gcc gac aag cag aag aac ggcatc aag gtg aac ttc aag atc 5034 Ile Met Ala Asp Lys Gln Lys Asn Gly IleLys Val Asn Phe Lys Ile 155 160 165 cgc cac aac atc gag gac ggc agc gtgcag ctc gcc gac cac tac cag 5082 Arg His Asn Ile Glu Asp Gly Ser Val GlnLeu Ala Asp His Tyr Gln 170 175 180 cag aac acc ccc atc ggc gac ggc cccgtg ctg ctg ccc gac aac cac 5130 Gln Asn Thr Pro Ile Gly Asp Gly Pro ValLeu Leu Pro Asp Asn His 185 190 195 200 tac ctg agc acc cag tcc gcc ctgagc aaa gac ccc aac gag aag cgc 5178 Tyr Leu Ser Thr Gln Ser Ala Leu SerLys Asp Pro Asn Glu Lys Arg 205 210 215 gat cac atg gtc ctg ctg gag ttcgtg acc gcc gcc ggg atc act ctc 5226 Asp His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly Ile Thr Leu 220 225 230 ggc atg gac gag ctg tac aag ggtaccacaagtt tgtacaaaaa agctgaacga 5280 Gly Met Asp Glu Leu Tyr Lys 235gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact 5340acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta ggcacccctt 5400aggaattcgc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg 5460tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggtg tgcttctaga 5520atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta 5580cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt 5640ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc 5700tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg 5760gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga 5820atatagaatt cctcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt 5880agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc 5940tcgttcagct ttcttgtaca aagtggtctc gagttaatta attgatccgg gttattagta 6000catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt 6060taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt 6120ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt aaaataggtt 6180tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat ctaatggatt 6240ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt 6300cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat gcgcttttgt 6360atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt taaataaagc 6420ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt cgtcgtccca 6480accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac gacgcctatt 6540aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg gaattatttc 6600tgattgcggg cgtttttggg cgggtttcaa tctaactgtg cccgatttta attcagacaa 6660cacgttagaa agcgatggtg caggcggtgg taacatttca gacggcaaat ctactaatgg 6720cggcggtggt ggagctgatg ataaatctac catcggtgga ggcgcaggcg gggctggcgg 6780cggaggcgga ggcggaggtg gtggcggtga tgcagacggc ggtttaggct caaatgtctc 6840tttaggcaac acagtcggca cctcaactat tgtactggtt tcgggcgccg tttttggttt 6900gaccggtctg agacgagtgc gatttttttc gtttctaata gcttccaaca attgttgtct 6960gtcgtctaaa ggtgcagcgg gttgaggttc cgtcggcatt ggtggagcgg gcggcaattc 7020agacatcgat ggtggtggtg gtggtggagg cgctggaatg ttaggcacgg gagaaggtgg 7080tggcggcggt gccgccggta taatttgttc tggtttagtt tgttcgcgca cgattgtggg 7140caccggcgca ggcgccgctg gctgcacaac ggaaggtcgt ctgcttcgag gcagcgcttg 7200gggtggtggc aattcaatat tataattgga atacaaatcg taaaaatctg ctataagcat 7260tgtaatttcg ctatcgttta ccgtgccgat atttaacaac cgctcaatgt aagcaattgt 7320attgtaaaga gattgtctca agctcggatc ccgcacgccg ataacaagcc ttttcatttt 7380tactacagca ttgtagtggc gagacacttc gctgtcgtcg acgtacatgt atgctttgtt 7440gtcaaaaacg tcgttggcaa gctttaaaat atttaaaaga acatctctgt tcagcaccac 7500tgtgttgtcg taaatgttgt ttttgataat ttgcgcttcc gcagtatcga cacgttcaaa 7560aaattgatgc gcatcaattt tgttgttcct attattgaat aaataagatt gtacagattc 7620atatctacga ttcgtcatgg ccaccacaaa tgctacgctg caaacgctgg tacaatttta 7680cgaaaactgc aaaaacgtca aaactcggta taaaataatc aacgggcgct ttggcaaaat 7740atctatttta tcgcacaagc ccactagcaa attgtatttg cagaaaacaa tttcggcgca 7800caattttaac gctgacgaaa taaaagttca ccagttaatg agcgaccacc caaattttat 7860aaaaatctat tttaatcacg gttccatcaa caaccaagtg atcgtgatgg actacattga 7920ctgtcccgat ttatttgaaa cactacaaat taaaggcgag ctttcgtacc aacttgttag 7980caatattatt agacagctgt gtgaagcgct caacgatttg cacaagcaca atttcataca 8040caacgacata aaactcgaaa atgtcttata tttcgaagca cttgatcgcg tgtatgtttg 8100cgattacgga ttgtgcaaac acgaaaactc acttagcgtg cacgacggca cgttggagta 8160ttttagtccg gaaaaaattc gacacacaac tatgcacgtt tcgtttgact ggtacgccgt 8220cggcgtgtta acatacaagt tgctaaccgg cggccgacac ccatttgaaa aaagcgaaga 8280cgaaatgttg gacttgaata gcatgaagcg tcgtcagcaa tacaatgaca ttggcgtttt 8340aaaacacgtt cgtaacgtta acgctcgtga ctttgtgtac tgcctaacaa gatacaacat 8400agattgtaga ctcacaaatt acaaacaaat tataaaacat gagtttttgt cgtaaaaatg 8460ccacttgttt tacgagtaga attctacgtg taacacacga tctaaaagat gatgtcattt 8520tttatcaatg actcatttgt tttaaaacag acttgtttta cgagtagaat tctacgtgta 8580aagcatgatc gtgagtggtg ttaataaaat cataaaaatt attgtaaatg tttattattt 8640aaaaacgatt caaatatata ataaaaacaa tctacatcta tttcttcaca atccataaca 8700cacaacaggt ccatcaatga gtttttgtct ttatccgaca tactatgtgc atgtaacaaa 8760tcaaatacat cttttaaatt tttatacaca tctttacatt gtctaccaaa atctttaata 8820accctataac aaggaaaaga cttttcttct tgcgtggttt tgccgcgcag atattgaaat 8880aaaatgtgca tgcacgacaa cttgtgttta ctaaaatgct ccttgcctat accgcaaaac 8940cggccataca tttcggcgat tacacgcgga caattgtacg attcgtctac gtgtaaacga 9000tcatcataat cactcttgcg caaacgaata aattttttca ccgcttccga caaacgaggc 9060accaattcgg cgggcacgct tcgatacatt attctgtgca cataagttac cacacaaaat 9120ttattgtacc accatccgac aacgtcgtta ttagggttga acacgttggc gatgcgcagc 9180agtttcccgt ttctcatgaa atattcaaag cggcccaaaa taatttgcaa gcaatccaac 9240atgtcttgag aaatttctcg ttcaaaattg ttcaaagaga atatctgcca tccgttttga 9300acgcgcacgc tgacgggaac caccgcatcg atttgctcca acacttcacg gacgttatcg 9360tcgatgccca tcgtttcgct ggtgctgaac caatgggaaa ggctcttgat ggaatcgccc 9420gcgtctatca tcttgaccgc ttcgtcaaag gtgcaactgc cgctcttcaa acgccgcata 9480gcggtcacgt cccgctctat gcacgacata ccgtttacgt acgattctga taggtattcc 9540tgaactatac ggtaatggtg atacgactcg ccatacacgt cgtgcacctc attgtattta 9600gcataataat tgtaaattat taactttgca gcgagagaca tgttgtcagt aaagcggtgc 9660taggctcaat aatactgatg tacaggcacg cgtgctattt atatataatt tcgcaaggag 9720gggagctgtt atcggttgct attattaaag aatggccgtc tgtttttatc acaagcttgg 9780cagcctcaac catgaagcgt cgtcattgta aattaaattc tctgcctcaa gaattatttg 9840acaagattgt cgagtattta tctttatctg attactgcaa tttggtgctt gtctgtaaaa 9900gaccttctag taaatataac gtgatatttg atagtactaa tcaccaacat ttgaaaggcg 9960tgtacaaaaa gacagacgtg caaataacaa gctacaacga 10000 6 21 DNA ArtificialSequence Baculo-Forward primer 6 actgttttcg taacagtttt g 21 7 19 DNAArtificial Sequence Baculo-Reverse primer 7 acaacgcaca gaatctagc 19 814000 DNA Artificial Sequence Polyhedrin locus of vBacHTS2 8 gaattctacccgtaaagcga gtttagtttt gaaaaacaaa tgacatcatt tgtataatga 60 catcatcccctgattgtgtt ttacaagtag aattctatcc gtaaagcgag ttcagttttg 120 aaaacaaatgagtcatacct aaacacgtta ataatcttct gatatcagct tatgactcaa 180 gttatgagccgtgtgcaaaa catgagataa gtttatgaca tcatccactg atcgtgcgtt 240 acaagtagaattctactcgt aaagccagtt cggttatgag ccgtgtgcaa aacatgacat 300 cagcttatgactcatacttg attgtgtttt acgcgtagaa ttctactcgt aaagcgagtt 360 cggttatgagccgtgtgcaa aacatgacat cagcttatga gtcataatta atcgtgcgtt 420 acaagtagaattctactcgt aaagcgagtt gaaggatcat atttagttgc gtttatgaga 480 taagattgaaagcacgtgta aaatgtttcc cgcgcgttgg cacaactatt tacaatgcgg 540 ccaagttataaaagattcta atctgatatg ttttaaaaca cctttgcggc ccgagttgtt 600 tgcgtacgtgactagcgaag aagatgtgtg gaccgcagaa cagatagtaa aacaaaaccc 660 tagtattggagcaataatcg atttaaccaa cacgtctaaa tattatgatg gtgtgcattt 720 tttgcgggcgggcctgttat acaaaaaaat tcaagtacct ggccagactt tgccgcctga 780 aagcatagttcaagaattta ttgacacggt aaaagaattt acagaaaagt gtcccggcat 840 gttggtgggcgtgcactgca cacacggtat taatcgcacc ggttacatgg tgtgcagata 900 tttaatgcacaccctgggta ttgcgccgca ggaagccata gatagattcg aaaaagccag 960 aggtcacaaaattgaaagac aaaattacgt tcaagattta ttaatttaat taatattatt 1020 tgcattctttaacaaatact ttatcctatt ttcaaattgt tgcgcttctt ccagcgaacc 1080 aaaactatgcttcgcttgct ccgtttagct tgtagccgat cagtggcgtt gttccaatcg 1140 acggtaggattaggccggat attctccacc acaatgttgg caacgttgat gttacgttta 1200 tgcttttggttttccacgta cgtcttttgg ccggtaatag ccgtaaacgt agtgccgtcg 1260 cgcgtcacgcacaacaccgg atgtttgcgc ttgtccgcgg ggtattgaac cgcgcgatcc 1320 gacaaatccaccactttggc aactaaatcg gtgacctgcg cgtctttttt ctgcattatt 1380 tcgtctttcttttgcatggt ttcctggaag ccggtgtaca tgcggtttag atcagtcatg 1440 acgcgcgtgacctgcaaatc tttggcctcg atctgcttgt ccttgatggc aacgatgcgt 1500 tcaataaactcttgtttttt aacaagttcc tcggtttttt gcgccaccac cgcttgcagc 1560 gcgtttgtgtgctcggtgaa tgtcgcaatc agcttagtca ccaactgttt gctctcctcc 1620 tcccgttgtttgatcgcggg atcgtacttg ccggtgcaga gcacttgagg aattacttct 1680 tctaaaagccattcttgtaa ttctatggcg taaggcaatt tggacttcat aatcagctga 1740 atcacgccggatttagtaat gagcactgta tgcggctgca aatacagcgg gtcgcccctt 1800 ttcacgacgctgttagaggt agggccccca ttttggatgg tctgctcaaa taacgatttg 1860 tatttattgtctacatgaac acgtatagct ttatcacaaa ctgtatattt taaactgtta 1920 gcgacgtccttggccacgaa ccggacctgt tggtcgcgct ctagcacgta ccgcaggttg 1980 aacgtatcttctccaaattt aaattctcca attttaacgc gagccatttt gatacacgtg 2040 tgtcgattttgcaacaacta ttgtttttta acgcaaacta aacttattgt ggtaagcaat 2100 aattaaatatgggggaacat gcgccgctac aacactcgtc gttatgaacg cagacggcgc 2160 cggtctcggcgcaagcggct aaaacgtgtt gcgcgttcaa cgcggcaaac atcgcaaaag 2220 ccaatagtacagttttgatt tgcatattaa cggcgatttt ttaaattatc ttatttaata 2280 aatagttatgacgcctacaa ctccccgccc gcgttgactc gctgcacctc gagcagttcg 2340 ttgacgccttcctccgtgtg gccgaacacg tcgagcgggt ggtcgatgac cagcggcgtg 2400 ccgcacgcgacgcacaagta tctgtacacc gaatgatcgt cgggcgaagg cacgtcggcc 2460 tccaagtggcaatattggca aattcgaaaa tatatacagt tgggttgttt gcgcatatct 2520 atcgtggcgttgggcatgta cgtccgaacg ttgatttgca tgcaagccga aattaaatca 2580 ttgcgattagtgcgattaaa acgttgtaca tcctcgcttt taatcatgcc gtcgattaaa 2640 tcgcgcaatcgagtcaagtg atcaaagtgt ggaataatgt tttctttgta ttcccgagtc 2700 aagcgcagcgcgtattttaa caaactagcc atcttgtaag ttagtttcat ttaatgcaac 2760 tttatccaataatatattat gtatcgcacg tcaagaatta acaatgcgcc cgttgtcgca 2820 tctcaacacgactatgatag agatcaaata aagcgcgaat taaatagctt gcgacgcaac 2880 gtgcacgatctgtgcacgcg ttccggcacg agctttgatt gtaataagtt tttacgaagc 2940 gatgacatgacccccgtagt gacaacgatc acgcccaaaa gaactgccga ctacaaaatt 3000 accgagtatgtcggtgacgt taaaactatt aagccatcca atcgaccgtt agtcgaatca 3060 ggaccgctggtgcgagaagc cgcgaagtat ggcgaatgca tcgtataacg tgtggagtcc 3120 gctcattagagcgtcatgtt tagacaagaa agctacatat ttaattgatc ccgatgattt 3180 tattgataaattgaccctaa ctccatacac ggtattctac aatggcgggg ttttggtcaa 3240 aatttccggactgcgattgt acatgctgtt aacggctccg cccactatta atgaaattaa 3300 aaattccaattttaaaaaac gcagcaagag aaacatttgt atgaaagaat gcgtagaagg 3360 aaagaaaaatgtcgtcgaca tgctgaacaa caagattaat atgcctccgt gtataaaaaa 3420 aatattgaacgatttgaaag aaaacaatgt accgcgcggc ggtatgtaca ggaagaggtt 3480 tatactaaactgttacattg caaacgtggt ttcgtgtgcc aagtgtgaaa accgatgttt 3540 aatcaaggctctgacgcatt tctacaacca cgactccaag tgtgtgggtg aagtcatgca 3600 tcttttaatcaaatcccaag atgtgtataa accaccaaac tgccaaaaaa tgaaaactgt 3660 cgacaagctctgtccgtttg ctggcaactg caagggtctc aatcctattt gtaattattg 3720 aataataaaacaattataaa tgctaaattt gttttttatt aacgatacaa accaaacgca 3780 acaagaacatttgtagtatt atctataatt gaaaacgcgt agttataatc gctgaggtaa 3840 tatttaaaatcattttcaaa tgattcacag ttaatttgcg acaatataat tttattttca 3900 cataaactagacgccttgtc gtcttcttct tcgtattcct tctctttttc atttttctcc 3960 tcataaaaattaacatagtt attatcgtat ccatatatgt atctatcgta tagagtaaat 4020 tttttgttgtcataaatata tatgtctttt ttaatggggt gtatagtacc gctgcgcata 4080 gtttttctgtaatttacaac agtgctattt tctggtagtt cttcggagtg tgttgcttta 4140 attattaaatttatataatc aatgaatttg ggatcgtcgg ttttgtacaa tatgttgccg 4200 gcatagtacgcagcttcttc tagttcaatt acaccatttt ttagcagcac cggattaaca 4260 taactttccaaaatgttgta cgaaccgtta aacaaaaaca gttcacctcc cttttctata 4320 ctattgtctgcgagcagttg tttgttgtta aaaataacag ccattgtaat gagacgcaca 4380 aactaatatcacaaactgga aatgtctatc aatatatagt tgctgatatc atggagataa 4440 ttaaaatgataaccatctcg caaataaata agtattttac tgttttcgta acagttttgt 4500 aataaaaaaacctataaata cggatcccgg ggtaccacaa gtttgtacaa aaaagctgaa 4560 cgagaaacgtaaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag 4620 actacataatactgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc 4680 cttaggtagggtcaccgtcg acagcgacac acttgcatcg gatgcagccc ggttaacgtg 4740 ccggcacggcctgggtaacc aggtattttg tccacataac cgtgcgcaaa atgttgtgga 4800 taagcaggacacagcagcaa tccacagcag gcatacaacc gcacaccgag gttactccgt 4860 tctacaggttacgacgacat gtcaatactt gcccttgaca ggcattgatg gaatcgtagt 4920 ctcacgctgatagtctgatc gacaatacaa gtgggaccgt ggtcccagac cgataatcag 4980 accgacaacacgagtgggat cgtggtccca gactaataat cagaccgacg atacgagtgg 5040 gaccgtggtcccagactaat aatcagaccg acgatacgag tgggaccgtg gttccagact 5100 aataatcagaccgacgatac gagtgggacc gtggtcccag actaataatc agaccgacga 5160 tacgagtgggaccatggtcc cagactaata atcagaccga cgatacgagt gggaccgtgg 5220 tcccagtctgattatcagac cgacgatacg agtgggaccg tggtcccaga ctaataatca 5280 gaccgacgatacgagtggga ccgtggtccc agactaataa tcagaccgac gatacgagtg 5340 ggaccgtggtcccagtctga ttatcagacc gacgatacaa gtggaacagt gggcccagag 5400 agaatattcaggccagttat gctttctggc ctgtaacaaa ggacattaag taaagacaga 5460 taaacgtagactaaaacgtg gtcgcatcag ggtgctggct tttcaagttc cttaagaatg 5520 gcctcaattttctctataca ctcagttgga acacgagacc tgtccaggtt aagcaccatt 5580 ttatcgcccttatacaatac tgtcgctcca ggagcaaact gatgtcgtga gcttaaacta 5640 gttcttgatgcagatgacgt tttaagcaca gaagttaaaa gagtgataac ttcttcagct 5700 tcaaatatcaccccagcttt tttctgctca tgaaggttag atgcctgctg cttaagtaat 5760 tcctctttatctgtaaaggc tttttgaagt gcatcacctg accgggcaga tagttcaccg 5820 gggtgagaaaaaagagcaac aactgattta ggcaatttgg cggtgttgat acagcgggta 5880 ataatcttacgtgaaatatt ttccgcatca gccagcgcag aaatatttcc agcaaattca 5940 ttctgcaatcggcttgcata acgctgacca cgttcataag cacttgttgg gcgataatcg 6000 ttacccaatctggataatgc agccatctgc tcatcatcca gctcgccaac cagaacacga 6060 taatcactttcggtaagtgc agcagcttta cgacggcgac tcccatcggc aatttctatg 6120 acaccagatactcttcgacc gaacgccggt gtctgttgac cagtcagtag aaaagaaggg 6180 atgagatcatccagtgcgtc ctcagtaagc agctcctggt cacgttcatt acctgaccat 6240 acccgagaggtcttctcaac actatcaccc cggagcactt caagagtaaa cttcacatcc 6300 cgaccacatacaggcaaagt aatggcatta ccgcgagcca ttactcctac gcgcgcaatt 6360 aacgaatccaccatcggggc agctggtgtc gataacgaag tatcttcaac cggttgagta 6420 ttgagcgtatgttttggaat aacaggcgca cgcttcatta tctaatctcc cagcgtggtt 6480 taatcagacgatcgaaaatt tcattgcaga caggttccca aatagaaaga gcatttctcc 6540 aggcaccagttgaagagcgt tgatcaatgg cctgttcaaa aacagttctc atccggatct 6600 gacctttaccaacttcatcc gtttcacgta caacattttt tagaaccatg cttccccagg 6660 catcccgaatttgctcctcc atccacgggg actgagagcc attactattg ctgtatttgg 6720 taagcaaaatacgtacatca ggctcgaacc ctttaagatc aacgttcttg agcagatcac 6780 gaagcatatcgaaaaactgc agtgcggagg tgtagtcaaa caactcagca ggcgtgggaa 6840 caatcagcacatcagcagca catacgacat taatcgtgcc gatacccagg ttaggcgcgc 6900 tgtcaataactatgacatca tagtcatgag caacagtttc aatggccagt cggagcatca 6960 ggtgtggatcggtgggcagt ttaccttcat caaatttgcc cattaactca gtttcaatac 7020 ggtgcagagccagacaggaa ggaataatgt caagccccgg ccagcaagtg ggctttattg 7080 cataagtgacatcgtccttt tccccaagat agaaaggcag gagagtgtct tctgcatgaa 7140 tatgaagatctggtacccat ccgtgataca ttgaggctgt tccctggggg tcgttacctt 7200 ccacgagcaaaacacgtagc cccttcagag ccagatcctg agcaagatga acagaaactg 7260 aggttttgtaaacgccacct ttatgggcag caaccccgat caccggtgga aatacgtctt 7320 cagcacgtcgcaatcgcgta ccaaacacat cacgcatatg attaatttgt tcaattgtat 7380 aaccaacacgttgctcaacc cgtcctcgaa tttccatatc cgggtgcggt agtcgccctg 7440 ctttctcggcatctctgata gcctgagaag aaaccccaac taaatccgct gcttcaccta 7500 ttctccagcgccgggttatt ttcctcgctt ccgggctgtc atcattaaac tgtgcaatgg 7560 cgatagccttcgtcatttca tgaccagcgt ttatgcactg gttaagtgtt tccatgagtt 7620 tcattctgaacatcctttaa tcattgcttt gcgttttttt attaaatctt gcaatttact 7680 gcaaagcaacaacaaaatcg caaagtcatc aaaaaaccgc aaagttgttt aaaataagag 7740 caacactacaaaaggagata agaagagcac atacctcagt cacttattat cactagcgct 7800 cgccgcagccgtgtaaccga gcatagcgag cgaactggcg aggaagcaaa gaagaactgt 7860 tctgtcagatagctcttacg ctcagcgcaa gaagaaatat ccaccgtggg aaaaactcca 7920 ggtagaggtacacacgcgga tagccaattc agagtaataa actgtgataa tcaaccctca 7980 tcaatgatgacgaactaacc cccgatatca ggtcacatga cgaagggaaa gagaaggaaa 8040 tcaactgtgacaaactgccc tcaaatttgg cttccttaaa aattacagtt caaaaagtat 8100 gagaaaatccatgcaggctg aaggaaacag caaaactgtg acaaattacc ctcagtaggt 8160 cagaacaaatgtgacgaacc accctcaaat ctgtgacaga taaccctcag actatcctgt 8220 cgtcatggaagtgatatcgc ggaaggaaaa tacgatatga gtcgtctggc ggcctttctt 8280 tttctcaatgtatgagaggc gcattggagt tctgctgttg atctcattaa cacagacctg 8340 caggaagcggcggcggaagt caggcatacg ctggtaactt tgaggcagct ggtaacgctc 8400 tatgatccagtcgattttca gagagacgat gcctgagcca tccggcttac gatactgaca 8460 cagggattcgtataaacgca tggcatacgg attggtgatt tcttttgttt cactaagccg 8520 aaactgcgtaaaccggttct gtaacccgat aaagaaggga atgagatatg ggttgatatg 8580 tacactgtaaagccctctgg atggactgtg cgcacgtttg ataaaccaag gaaaagattc 8640 atagcctttttcatcgccgg catcctcttc agggcgataa aaaaccactt ccttccccgc 8700 gaaactcttcaatgcctgcc gtatatcctt actggcttcc gcagaggtca atccgaatat 8760 ttcagcatatttagcaacat ggatctcgca gataccgtca tgttcctgta gggtgccatc 8820 agattttctgatctggtcaa cgaacagata cagcatacgt ttttgatccc gggagagact 8880 atatgccgcctcagtgaggt cgtttgactg gacgattcgc gggctatttt tacgtttctt 8940 gtgattgataaccgctgttt ccgccatgac agatccatgt gaagtgtgac aagtttttag 9000 attgtcacactaaataaaaa agagtcaata agcagggata actttgtgaa aaaacagctt 9060 cttctgagggcaatttgtca cagggttaag ggcaatttgt cacagacagg actgtcattt 9120 gagggtgatttgtcacactg aaagggcaat ttgtcacaac accttctcta gaaccagcat 9180 ggataaaggcctacaaggcg ctctaaaaaa gaagatctaa aaactataaa aaaaataatt 9240 ataaaaatatccccgtggat aagtggataa ccccaaggga agttttttca ggcatcgtgt 9300 gtaagcagaatatataagtg ctgttccctg gtgcttcctc gctcactcga gggcttcgcc 9360 ctgtcgctcaactgcggcga gcactactgg ctgtaaaagg acagaccaca tcatggttct 9420 gtgttcattaggttgttctg tccattgctg acataatccg ctccacttca acgtaacacc 9480 gcacgaagatttctattgtt cctgaaggca tattcaaatc gttttcgtta ccgcttgcag 9540 gcatcatgacagaacactac ttcctataaa cgctacacag gctcctgaga ttaataatgc 9600 ggatctctacgataatggga gattttcccg actgtttcgt tcgcttctca gtggataaca 9660 gccagcttctctgtttaaca gacaaaaaca gcatatccac tcagttccac atttccatat 9720 aaaggccaaggcatttattc tcaggataat tgtttcagca tcgcaaccgc atcagactcc 9780 ggcatcgcaaactgcacccg gtgccgggca gccacatcca gcgcaaaaac cttcgtgtag 9840 acttccgttgaactgatgga cttatgtccc atcaggcttt gcagaacttt cagcggtata 9900 ccggcatacagcatgtgcat cgcataggaa tggcggaacg tatgtggtgt gaccggaaca 9960 gagaacgtcacaccgtcagc agcagcggcg gcaaccgcct ccccaatcca ggtcctgacc 10020 gttctgtccgtcacttccca gatccgcgct ttctctgtcc ttcctgtgcg acggttacgc 10080 cgctccatgagcttatcgcg aataaatacc tgtgacggaa gatcacttcg cagaataaat 10140 aaatcctggtgtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga 10200 cgttgatcggcacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg 10260 cgtattttttgagttatcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat 10320 cactggatataccaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt 10380 tcagtcagttgctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt 10440 aaagaccgtaaagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg 10500 cctgatgaatgctcatccgg agttccgtat ggcaatgaaa gacggtgagc tggtgatatg 10560 ggatagtgttcacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct 10620 ctggagtgaataccacgacg atttccggca gtttctacac atatattcgc aagatgtggc 10680 gtgttacggtgaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt 10740 ctcagccaatccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa 10800 cttcttcgcccccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat 10860 gccgctggcgattcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct 10920 taatgaattacaacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag 10980 ttattggtgcccttaaacgc ctggttgcta cgcctgaata agtgataata agcggatgaa 11040 tggcagaaattcgatgataa gctgtcaaac atgagaattg gtcgacggcg cgccaaagct 11100 tgcatgcctgcagccgcgta acctggcaaa atcggttacg gttgagtaat aaatggatgc 11160 cctgcgtaagcggggcacat ttcattacct ctttctccgc acccgaccct caggtcgacc 11220 atagtgactggatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct 11280 aatttaatatattgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 11340 tggtctcgagttaattaatt gatccgggtt attagtacat ttattaagcg ctagattctg 11400 tgcgttgttgatttacagac aattgttgta cgtattttaa taattcatta aatttataat 11460 ctttagggtggtatgttaga gcgaaaatca aatgattttc agcgtcttta tatctgaatt 11520 taaatattaaatcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt 11580 gtttttccgaaccgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg 11640 ccaaatcttgtagcagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata 11700 aaggttcgacgtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag 11760 tgtacaattgactcgacgta aacacgttaa ataaagcttg gacatattta acatcgggcg 11820 tgttagctttattaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt 11880 ccgaagacgattttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg 11940 cgatcaaatttgtagttgag ctttttggaa ttatttctga ttgcgggcgt ttttgggcgg 12000 gtttcaatctaactgtgccc gattttaatt cagacaacac gttagaaagc gatggtgcag 12060 gcggtggtaacatttcagac ggcaaatcta ctaatggcgg cggtggtgga gctgatgata 12120 aatctaccatcggtggaggc gcaggcgggg ctggcggcgg aggcggaggc ggaggtggtg 12180 gcggtgatgcagacggcggt ttaggctcaa atgtctcttt aggcaacaca gtcggcacct 12240 caactattgtactggtttcg ggcgccgttt ttggtttgac cggtctgaga cgagtgcgat 12300 ttttttcgtttctaatagct tccaacaatt gttgtctgtc gtctaaaggt gcagcgggtt 12360 gaggttccgtcggcattggt ggagcgggcg gcaattcaga catcgatggt ggtggtggtg 12420 gtggaggcgctggaatgtta ggcacgggag aaggtggtgg cggcggtgcc gccggtataa 12480 tttgttctggtttagtttgt tcgcgcacga ttgtgggcac cggcgcaggc gccgctggct 12540 gcacaacggaaggtcgtctg cttcgaggca gcgcttgggg tggtggcaat tcaatattat 12600 aattggaatacaaatcgtaa aaatctgcta taagcattgt aatttcgcta tcgtttaccg 12660 tgccgatatttaacaaccgc tcaatgtaag caattgtatt gtaaagagat tgtctcaagc 12720 tcggatcccgcacgccgata acaagccttt tcatttttac tacagcattg tagtggcgag 12780 acacttcgctgtcgtcgacg tacatgtatg ctttgttgtc aaaaacgtcg ttggcaagct 12840 ttaaaatatttaaaagaaca tctctgttca gcaccactgt gttgtcgtaa atgttgtttt 12900 tgataatttgcgcttccgca gtatcgacac gttcaaaaaa ttgatgcgca tcaattttgt 12960 tgttcctattattgaataaa taagattgta cagattcata tctacgattc gtcatggcca 13020 ccacaaatgctacgctgcaa acgctggtac aattttacga aaactgcaaa aacgtcaaaa 13080 ctcggtataaaataatcaac gggcgctttg gcaaaatatc tattttatcg cacaagccca 13140 ctagcaaattgtatttgcag aaaacaattt cggcgcacaa ttttaacgct gacgaaataa 13200 aagttcaccagttaatgagc gaccacccaa attttataaa aatctatttt aatcacggtt 13260 ccatcaacaaccaagtgatc gtgatggact acattgactg tcccgattta tttgaaacac 13320 tacaaattaaaggcgagctt tcgtaccaac ttgttagcaa tattattaga cagctgtgtg 13380 aagcgctcaacgatttgcac aagcacaatt tcatacacaa cgacataaaa ctcgaaaatg 13440 tcttatatttcgaagcactt gatcgcgtgt atgtttgcga ttacggattg tgcaaacacg 13500 aaaactcacttagcgtgcac gacggcacgt tggagtattt tagtccggaa aaaattcgac 13560 acacaactatgcacgtttcg tttgactggt acgccgtcgg cgtgttaaca tacaagttgc 13620 taaccggcggccgacaccca tttgaaaaaa gcgaagacga aatgttggac ttgaatagca 13680 tgaagcgtcgtcagcaatac aatgacattg gcgttttaaa acacgttcgt aacgttaacg 13740 ctcgtgactttgtgtactgc ctaacaagat acaacataga ttgtagactc acaaattaca 13800 aacaaattataaaacatgag tttttgtcgt aaaaatgcca cttgttttac gagtagaatt 13860 ctacgtgtaacacacgatct aaaagatgat gtcatttttt atcaatgact catttgtttt 13920 aaacagacttgttttacga gtagaattct acgtgtaaag catgatcgtg agtggtgtta 13980 taaaatcataaaaattatt 14000 9 13100 DNA Artificial Sequence Polyhedrin locus ofvBacHTS2_GFP 9 gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa tgacatcatttgtataatga 60 catcatcccc tgattgtgtt ttacaagtag aattctatcc gtaaagcgagttcagttttg 120 aaaacaaatg agtcatacct aaacacgtta ataatcttct gatatcagcttatgactcaa 180 gttatgagcc gtgtgcaaaa catgagataa gtttatgaca tcatccactgatcgtgcgtt 240 acaagtagaa ttctactcgt aaagccagtt cggttatgag ccgtgtgcaaaacatgacat 300 cagcttatga ctcatacttg attgtgtttt acgcgtagaa ttctactcgtaaagcgagtt 360 cggttatgag ccgtgtgcaa aacatgacat cagcttatga gtcataattaatcgtgcgtt 420 acaagtagaa ttctactcgt aaagcgagtt gaaggatcat atttagttgcgtttatgaga 480 taagattgaa agcacgtgta aaatgtttcc cgcgcgttgg cacaactatttacaatgcgg 540 ccaagttata aaagattcta atctgatatg ttttaaaaca cctttgcggcccgagttgtt 600 tgcgtacgtg actagcgaag aagatgtgtg gaccgcagaa cagatagtaaaacaaaaccc 660 tagtattgga gcaataatcg atttaaccaa cacgtctaaa tattatgatggtgtgcattt 720 tttgcgggcg ggcctgttat acaaaaaaat tcaagtacct ggccagactttgccgcctga 780 aagcatagtt caagaattta ttgacacggt aaaagaattt acagaaaagtgtcccggcat 840 gttggtgggc gtgcactgca cacacggtat taatcgcacc ggttacatggtgtgcagata 900 tttaatgcac accctgggta ttgcgccgca ggaagccata gatagattcgaaaaagccag 960 aggtcacaaa attgaaagac aaaattacgt tcaagattta ttaatttaattaatattatt 1020 tgcattcttt aacaaatact ttatcctatt ttcaaattgt tgcgcttcttccagcgaacc 1080 aaaactatgc ttcgcttgct ccgtttagct tgtagccgat cagtggcgttgttccaatcg 1140 acggtaggat taggccggat attctccacc acaatgttgg caacgttgatgttacgttta 1200 tgcttttggt tttccacgta cgtcttttgg ccggtaatag ccgtaaacgtagtgccgtcg 1260 cgcgtcacgc acaacaccgg atgtttgcgc ttgtccgcgg ggtattgaaccgcgcgatcc 1320 gacaaatcca ccactttggc aactaaatcg gtgacctgcg cgtcttttttctgcattatt 1380 tcgtctttct tttgcatggt ttcctggaag ccggtgtaca tgcggtttagatcagtcatg 1440 acgcgcgtga cctgcaaatc tttggcctcg atctgcttgt ccttgatggcaacgatgcgt 1500 tcaataaact cttgtttttt aacaagttcc tcggtttttt gcgccaccaccgcttgcagc 1560 gcgtttgtgt gctcggtgaa tgtcgcaatc agcttagtca ccaactgtttgctctcctcc 1620 tcccgttgtt tgatcgcggg atcgtacttg ccggtgcaga gcacttgaggaattacttct 1680 tctaaaagcc attcttgtaa ttctatggcg taaggcaatt tggacttcataatcagctga 1740 atcacgccgg atttagtaat gagcactgta tgcggctgca aatacagcgggtcgcccctt 1800 ttcacgacgc tgttagaggt agggccccca ttttggatgg tctgctcaaataacgatttg 1860 tatttattgt ctacatgaac acgtatagct ttatcacaaa ctgtatattttaaactgtta 1920 gcgacgtcct tggccacgaa ccggacctgt tggtcgcgct ctagcacgtaccgcaggttg 1980 aacgtatctt ctccaaattt aaattctcca attttaacgc gagccattttgatacacgtg 2040 tgtcgatttt gcaacaacta ttgtttttta acgcaaacta aacttattgtggtaagcaat 2100 aattaaatat gggggaacat gcgccgctac aacactcgtc gttatgaacgcagacggcgc 2160 cggtctcggc gcaagcggct aaaacgtgtt gcgcgttcaa cgcggcaaacatcgcaaaag 2220 ccaatagtac agttttgatt tgcatattaa cggcgatttt ttaaattatcttatttaata 2280 aatagttatg acgcctacaa ctccccgccc gcgttgactc gctgcacctcgagcagttcg 2340 ttgacgcctt cctccgtgtg gccgaacacg tcgagcgggt ggtcgatgaccagcggcgtg 2400 ccgcacgcga cgcacaagta tctgtacacc gaatgatcgt cgggcgaaggcacgtcggcc 2460 tccaagtggc aatattggca aattcgaaaa tatatacagt tgggttgtttgcgcatatct 2520 atcgtggcgt tgggcatgta cgtccgaacg ttgatttgca tgcaagccgaaattaaatca 2580 ttgcgattag tgcgattaaa acgttgtaca tcctcgcttt taatcatgccgtcgattaaa 2640 tcgcgcaatc gagtcaagtg atcaaagtgt ggaataatgt tttctttgtattcccgagtc 2700 aagcgcagcg cgtattttaa caaactagcc atcttgtaag ttagtttcatttaatgcaac 2760 tttatccaat aatatattat gtatcgcacg tcaagaatta acaatgcgcccgttgtcgca 2820 tctcaacacg actatgatag agatcaaata aagcgcgaat taaatagcttgcgacgcaac 2880 gtgcacgatc tgtgcacgcg ttccggcacg agctttgatt gtaataagtttttacgaagc 2940 gatgacatga cccccgtagt gacaacgatc acgcccaaaa gaactgccgactacaaaatt 3000 accgagtatg tcggtgacgt taaaactatt aagccatcca atcgaccgttagtcgaatca 3060 ggaccgctgg tgcgagaagc cgcgaagtat ggcgaatgca tcgtataacgtgtggagtcc 3120 gctcattaga gcgtcatgtt tagacaagaa agctacatat ttaattgatcccgatgattt 3180 tattgataaa ttgaccctaa ctccatacac ggtattctac aatggcggggttttggtcaa 3240 aatttccgga ctgcgattgt acatgctgtt aacggctccg cccactattaatgaaattaa 3300 aaattccaat tttaaaaaac gcagcaagag aaacatttgt atgaaagaatgcgtagaagg 3360 aaagaaaaat gtcgtcgaca tgctgaacaa caagattaat atgcctccgtgtataaaaaa 3420 aatattgaac gatttgaaag aaaacaatgt accgcgcggc ggtatgtacaggaagaggtt 3480 tatactaaac tgttacattg caaacgtggt ttcgtgtgcc aagtgtgaaaaccgatgttt 3540 aatcaaggct ctgacgcatt tctacaacca cgactccaag tgtgtgggtgaagtcatgca 3600 tcttttaatc aaatcccaag atgtgtataa accaccaaac tgccaaaaaatgaaaactgt 3660 cgacaagctc tgtccgtttg ctggcaactg caagggtctc aatcctatttgtaattattg 3720 aataataaaa caattataaa tgctaaattt gttttttatt aacgatacaaaccaaacgca 3780 acaagaacat ttgtagtatt atctataatt gaaaacgcgt agttataatcgctgaggtaa 3840 tatttaaaat cattttcaaa tgattcacag ttaatttgcg acaatataattttattttca 3900 cataaactag acgccttgtc gtcttcttct tcgtattcct tctctttttcatttttctcc 3960 tcataaaaat taacatagtt attatcgtat ccatatatgt atctatcgtatagagtaaat 4020 tttttgttgt cataaatata tatgtctttt ttaatggggt gtatagtaccgctgcgcata 4080 gtttttctgt aatttacaac agtgctattt tctggtagtt cttcggagtgtgttgcttta 4140 attattaaat ttatataatc aatgaatttg ggatcgtcgg ttttgtacaatatgttgccg 4200 gcatagtacg cagcttcttc tagttcaatt acaccatttt ttagcagcaccggattaaca 4260 taactttcca aaatgttgta cgaaccgtta aacaaaaaca gttcacctcccttttctata 4320 ctattgtctg cgagcagttg tttgttgtta aaaataacag ccattgtaatgagacgcaca 4380 aactaatatc acaaactgga aatgtctatc aatatatagt tgctgatatcatggagataa 4440 ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgtaacagttttgt 4500 aataaaaaaa cctataaata cggatccacc atg gtg agc aag ggc gaggag ctg 4554 Met Val Ser Lys Gly Glu Glu Leu 1 5 ttc acc ggg gtg gtg cccatc ctg gtc gag ctg gac ggc gac gta aac 4602 Phe Thr Gly Val Val Pro IleLeu Val Glu Leu Asp Gly Asp Val Asn 10 15 20 ggc cac aag ttc agc gtg tccggc gag ggc gag ggc gat gcc acc tac 4650 Gly His Lys Phe Ser Val Ser GlyGlu Gly Glu Gly Asp Ala Thr Tyr 25 30 35 40 ggc aag ctg acc ctg aag ttcatc tgc acc acc ggc aag ctg ccc gtg 4698 Gly Lys Leu Thr Leu Lys Phe IleCys Thr Thr Gly Lys Leu Pro Val 45 50 55 ccc tgg ccc acc ctc gtg acc accctg acc tgg ggc gtg cag tgc ttc 4746 Pro Trp Pro Thr Leu Val Thr Thr LeuThr Trp Gly Val Gln Cys Phe 60 65 70 agc cgc tac ccc gac cac atg aag cagcac gac ttc ttc aag tcc gcc 4794 Ser Arg Tyr Pro Asp His Met Lys Gln HisAsp Phe Phe Lys Ser Ala 75 80 85 atg ccc gaa ggc tac gtc cag gag cgc accatc ttc ttc aag gac gac 4842 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr IlePhe Phe Lys Asp Asp 90 95 100 ggc aac tac aag acc cgc gcc gag gtg aagttc gag ggc gac acc ctg 4890 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys PheGlu Gly Asp Thr Leu 105 110 115 120 gtg aac cgc atc gag ctg aag ggc atcgac ttc aag gag gac ggc aac 4938 Val Asn Arg Ile Glu Leu Lys Gly Ile AspPhe Lys Glu Asp Gly Asn 125 130 135 atc ctg ggg cac aag ctg gag tac aactac aac agc cac aac gtc tat 4986 Ile Leu Gly His Lys Leu Glu Tyr Asn TyrAsn Ser His Asn Val Tyr 140 145 150 atc atg gcc gac aag cag aag aac ggcatc aag gtg aac ttc aag atc 5034 Ile Met Ala Asp Lys Gln Lys Asn Gly IleLys Val Asn Phe Lys Ile 155 160 165 cgc cac aac atc gag gac ggc agc gtgcag ctc gcc gac cac tac cag 5082 Arg His Asn Ile Glu Asp Gly Ser Val GlnLeu Ala Asp His Tyr Gln 170 175 180 cag aac acc ccc atc ggc gac ggc cccgtg ctg ctg ccc gac aac cac 5130 Gln Asn Thr Pro Ile Gly Asp Gly Pro ValLeu Leu Pro Asp Asn His 185 190 195 200 tac ctg agc acc cag tcc gcc ctgagc aaa gac ccc aac gag aag cgc 5178 Tyr Leu Ser Thr Gln Ser Ala Leu SerLys Asp Pro Asn Glu Lys Arg 205 210 215 gat cac atg gtc ctg ctg gag ttcgtg acc gcc gcc ggg atc act ctc 5226 Asp His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly Ile Thr Leu 220 225 230 ggc atg gac gag ctg tac aag ggtaccacaagtt tgtacaaaaa agctgaacga 5280 Gly Met Asp Glu Leu Tyr Lys 235gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact 5340acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta ggcacccctt 5400aggtagggtc accgtcgaca gcgacacact tgcatcggat gcagcccggt taacgtgccg 5460gcacggcctg ggtaaccagg tattttgtcc acataaccgt gcgcaaaatg ttgtggataa 5520gcaggacaca gcagcaatcc acagcaggca tacaaccgca caccgaggtt actccgttct 5580acaggttacg acgacatgtc aatacttgcc cttgacaggc attgatggaa tcgtagtctc 5640acgctgatag tctgatcgac aatacaagtg ggaccgtggt cccagaccga taatcagacc 5700gacaacacga gtgggatcgt ggtcccagac taataatcag accgacgata cgagtgggac 5760cgtggtccca gactaataat cagaccgacg atacgagtgg gaccgtggtt ccagactaat 5820aatcagaccg acgatacgag tgggaccgtg gtcccagact aataatcaga ccgacgatac 5880gagtgggacc atggtcccag actaataatc agaccgacga tacgagtggg accgtggtcc 5940cagtctgatt atcagaccga cgatacgagt gggaccgtgg tcccagacta ataatcagac 6000cgacgatacg agtgggaccg tggtcccaga ctaataatca gaccgacgat acgagtggga 6060ccgtggtccc agtctgatta tcagaccgac gatacaagtg gaacagtggg cccagagaga 6120atattcaggc cagttatgct ttctggcctg taacaaagga cattaagtaa agacagataa 6180acgtagacta aaacgtggtc gcatcagggt gctggctttt caagttcctt aagaatggcc 6240tcaattttct ctatacactc agttggaaca cgagacctgt ccaggttaag caccatttta 6300tcgcccttat acaatactgt cgctccagga gcaaactgat gtcgtgagct taaactagtt 6360cttgatgcag atgacgtttt aagcacagaa gttaaaagag tgataacttc ttcagcttca 6420aatatcaccc cagctttttt ctgctcatga aggttagatg cctgctgctt aagtaattcc 6480tctttatctg taaaggcttt ttgaagtgca tcacctgacc gggcagatag ttcaccgggg 6540tgagaaaaaa gagcaacaac tgatttaggc aatttggcgg tgttgataca gcgggtaata 6600atcttacgtg aaatattttc cgcatcagcc agcgcagaaa tatttccagc aaattcattc 6660tgcaatcggc ttgcataacg ctgaccacgt tcataagcac ttgttgggcg ataatcgtta 6720cccaatctgg ataatgcagc catctgctca tcatccagct cgccaaccag aacacgataa 6780tcactttcgg taagtgcagc agctttacga cggcgactcc catcggcaat ttctatgaca 6840ccagatactc ttcgaccgaa cgccggtgtc tgttgaccag tcagtagaaa agaagggatg 6900agatcatcca gtgcgtcctc agtaagcagc tcctggtcac gttcattacc tgaccatacc 6960cgagaggtct tctcaacact atcaccccgg agcacttcaa gagtaaactt cacatcccga 7020ccacatacag gcaaagtaat ggcattaccg cgagccatta ctcctacgcg cgcaattaac 7080gaatccacca tcggggcagc tggtgtcgat aacgaagtat cttcaaccgg ttgagtattg 7140agcgtatgtt ttggaataac aggcgcacgc ttcattatct aatctcccag cgtggtttaa 7200tcagacgatc gaaaatttca ttgcagacag gttcccaaat agaaagagca tttctccagg 7260caccagttga agagcgttga tcaatggcct gttcaaaaac agttctcatc cggatctgac 7320ctttaccaac ttcatccgtt tcacgtacaa cattttttag aaccatgctt ccccaggcat 7380cccgaatttg ctcctccatc cacggggact gagagccatt actattgctg tatttggtaa 7440gcaaaatacg tacatcaggc tcgaaccctt taagatcaac gttcttgagc agatcacgaa 7500gcatatcgaa aaactgcagt gcggaggtgt agtcaaacaa ctcagcaggc gtgggaacaa 7560tcagcacatc agcagcacat acgacattaa tcgtgccgat acccaggtta ggcgcgctgt 7620caataactat gacatcatag tcatgagcaa cagtttcaat ggccagtcgg agcatcaggt 7680gtggatcggt gggcagttta ccttcatcaa atttgcccat taactcagtt tcaatacggt 7740gcagagccag acaggaagga ataatgtcaa gccccggcca gcaagtgggc tttattgcat 7800aagtgacatc gtccttttcc ccaagataga aaggcaggag agtgtcttct gcatgaatat 7860gaagatctgg tacccatccg tgatacattg aggctgttcc ctgggggtcg ttaccttcca 7920cgagcaaaac acgtagcccc ttcagagcca gatcctgagc aagatgaaca gaaactgagg 7980ttttgtaaac gccaccttta tgggcagcaa ccccgatcac cggtggaaat acgtcttcag 8040cacgtcgcaa tcgcgtacca aacacatcac gcatatgatt aatttgttca attgtataac 8100caacacgttg ctcaacccgt cctcgaattt ccatatccgg gtgcggtagt cgccctgctt 8160tctcggcatc tctgatagcc tgagaagaaa ccccaactaa atccgctgct tcacctattc 8220tccagcgccg ggttattttc ctcgcttccg ggctgtcatc attaaactgt gcaatggcga 8280tagccttcgt catttcatga ccagcgttta tgcactggtt aagtgtttcc atgagtttca 8340ttctgaacat cctttaatca ttgctttgcg tttttttatt aaatcttgca atttactgca 8400aagcaacaac aaaatcgcaa agtcatcaaa aaaccgcaaa gttgtttaaa ataagagcaa 8460cactacaaaa ggagataaga agagcacata cctcagtcac ttattatcac tagcgctcgc 8520cgcagccgtg taaccgagca tagcgagcga actggcgagg aagcaaagaa gaactgttct 8580gtcagatagc tcttacgctc agcgcaagaa gaaatatcca ccgtgggaaa aactccaggt 8640agaggtacac acgcggatag ccaattcaga gtaataaact gtgataatca accctcatca 8700atgatgacga actaaccccc gatatcaggt cacatgacga agggaaagag aaggaaatca 8760actgtgacaa actgccctca aatttggctt ccttaaaaat tacagttcaa aaagtatgag 8820aaaatccatg caggctgaag gaaacagcaa aactgtgaca aattaccctc agtaggtcag 8880aacaaatgtg acgaaccacc ctcaaatctg tgacagataa ccctcagact atcctgtcgt 8940catggaagtg atatcgcgga aggaaaatac gatatgagtc gtctggcggc ctttcttttt 9000ctcaatgtat gagaggcgca ttggagttct gctgttgatc tcattaacac agacctgcag 9060gaagcggcgg cggaagtcag gcatacgctg gtaactttga ggcagctggt aacgctctat 9120gatccagtcg attttcagag agacgatgcc tgagccatcc ggcttacgat actgacacag 9180ggattcgtat aaacgcatgg catacggatt ggtgatttct tttgtttcac taagccgaaa 9240ctgcgtaaac cggttctgta acccgataaa gaagggaatg agatatgggt tgatatgtac 9300actgtaaagc cctctggatg gactgtgcgc acgtttgata aaccaaggaa aagattcata 9360gcctttttca tcgccggcat cctcttcagg gcgataaaaa accacttcct tccccgcgaa 9420actcttcaat gcctgccgta tatccttact ggcttccgca gaggtcaatc cgaatatttc 9480agcatattta gcaacatgga tctcgcagat accgtcatgt tcctgtaggg tgccatcaga 9540ttttctgatc tggtcaacga acagatacag catacgtttt tgatcccggg agagactata 9600tgccgcctca gtgaggtcgt ttgactggac gattcgcggg ctatttttac gtttcttgtg 9660attgataacc gctgtttccg ccatgacaga tccatgtgaa gtgtgacaag tttttagatt 9720gtcacactaa ataaaaaaga gtcaataagc agggataact ttgtgaaaaa acagcttctt 9780ctgagggcaa tttgtcacag ggttaagggc aatttgtcac agacaggact gtcatttgag 9840ggtgatttgt cacactgaaa gggcaatttg tcacaacacc ttctctagaa ccagcatgga 9900taaaggccta caaggcgctc taaaaaagaa gatctaaaaa ctataaaaaa aataattata 9960aaaatatccc cgtggataag tggataaccc caagggaagt tttttcaggc atcgtgtgta 10020agcagaatat ataagtgctg ttccctggtg cttcctcgct cactcgaggg cttcgccctg 10080tcgctcaact gcggcgagca ctactggctg taaaaggaca gaccacatca tggttctgtg 10140ttcattaggt tgttctgtcc attgctgaca taatccgctc cacttcaacg taacaccgca 10200cgaagatttc tattgttcct gaaggcatat tcaaatcgtt ttcgttaccg cttgcaggca 10260tcatgacaga acactacttc ctataaacgc tacacaggct cctgagatta ataatgcgga 10320tctctacgat aatgggagat tttcccgact gtttcgttcg cttctcagtg gataacagcc 10380agcttctctg tttaacagac aaaaacagca tatccactca gttccacatt tccatataaa 10440ggccaaggca tttattctca ggataattgt ttcagcatcg caaccgcatc agactccggc 10500atcgcaaact gcacccggtg ccgggcagcc acatccagcg caaaaacctt cgtgtagact 10560tccgttgaac tgatggactt atgtcccatc aggctttgca gaactttcag cggtataccg 10620gcatacagca tgtgcatcgc ataggaatgg cggaacgtat gtggtgtgac cggaacagag 10680aacgtcacac cgtcagcagc agcggcggca accgcctccc caatccaggt cctgaccgtt 10740ctgtccgtca cttcccagat ccgcgctttc tctgtccttc ctgtgcgacg gttacgccgc 10800tccatgagct tatcgcgaat aaatacctgt gacggaagat cacttcgcag aataaataaa 10860tcctggtgtc cctgttgata ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt 10920tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt 10980attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac 11040tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca 11100gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa 11160gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct 11220gatgaatgct catccggagt tccgtatggc aatgaaagac ggtgagctgg tgatatggga 11280tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg 11340gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg 11400ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc 11460agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt 11520cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc 11580gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa 11640tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta 11700ttggtgccct taaacgcctg gttgctacgc ctgaataagt gataataagc ggatgaatgg 11760cagaaattcg atgataagct gtcaaacatg agaattggtc gacggcgcgc caaagcttgc 11820atgcctgcag ccgcgtaacc tggcaaaatc ggttacggtt gagtaataaa tggatgccct 11880gcgtaagcgg ggcacatttc attacctctt tctccgcacc cgaccctcag gtcgaccata 11940gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat 12000ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg 12060tctcgagtta attaattgat ccgggttatt agtacattta ttaagcgcta gattctgtgc 12120gttgttgatt tacagacaat tgttgtacgt attttaataa ttcattaaat ttataatctt 12180tagggtggta tgttagagcg aaaatcaaat gattttcagc gtctttatat ctgaatttaa 12240atattaaatc ctcaatagat ttgtaaaata ggtttcgatt agtttcaaac aagggttgtt 12300tttccgaacc gatggctgga ctatctaatg gattttcgct caacgccaca aaacttgcca 12360aatcttgtag cagcaatcta gctttgtcga tattcgtttg tgttttgttt tgtaataaag 12420gttcgacgtc gttcaaaata ttatgcgctt ttgtatttct ttcatcactg tcgttagtgt 12480acaattgact cgacgtaaac acgttaaata aagcttggac atatttaaca tcgggcgtgt 12540tagctttatt aggccgatta tcgtcgtcgt cccaaccctc gtcgttagaa gttgcttccg 12600aagacgattt tgccatagcc acacgacgcc tattaattgt gtcggctaac acgtccgcga 12660tcaaatttgt agttgagctt tttggaatta tttctgattg cgggcgtttt tgggcgggtt 12720tcaatctaac tgtgcccgat tttaattcag acaacacgtt agaaagcgat ggtgcaggcg 12780gtggtaacat ttcagacggc aaatctacta atggcggcgg tggtggagct gatgataaat 12840ctaccatcgg tggaggcgca ggcggggctg gcggcggagg cggaggcgga ggtggtggcg 12900gtgatgcaga cggcggttta ggctcaaatg tctctttagg caacacagtc ggcacctcaa 12960ctattgtact ggtttcgggc gccgtttttg gtttgaccgg tctgagacga gtgcgatttt 13020ttcgtttct aatagcttcc aacaattgtt gtctgtcgtc taaaggtgca gcgggttgag 13080ttccgtcgg cattggtgga 13100

What is claimed is:
 1. A method for preparing expressive circularrecombinant viral vectors, which comprises the steps of: i) providingviral genomic DNA containing at least two site-specific recombinationsites and at least one restriction enzyme recognition site; ii)providing gene cassette containing desired genomic materials flankedwith at least two site-specific sites; and iii) reacting DNA fragment ofi) and gene cassette of ii) in vitro.
 2. The method for preparingexpressive circular recombinant viral vectors as claimed in claim 1,wherein the viral genomic DNAs are digested with restriction enzymes atone or more sites to generate linear formed viral genomic DNAs.
 3. Themethod for preparing expressive circular recombinant viral vectors asclaimed in claim 2, wherein the restriction enzyme recognizes basesequences of CCTNAGG.
 4. The method for preparing expressive circularrecombinant viral vectors as claimed in claim 1, wherein the virus isbaculovirus, poliomavirus, papilomavirus, or HBV.
 5. A recombinant viralvector prepared according to any one of methods of claims 1 to
 4. 6. Amethod of screening recombinant viruses containing desired genomicmaterials, which comprises the steps of: i) providing viral genomic DNAfragment containing at least two site-specific recombination sites andat least one restriction enzyme recognition site; ii) providing genecassette containing desired genomic materials flanked with at least twosite-specific sites; iii) reacting DNA fragment of i) and gene cassetteof ii) in vitro to produce reaction mixture including expressivecircular recombinant viral vectors; iv) transferring the reactionmixture of the iii) to host cells in multi-well plate; and v)identifying the features expressed in host cells.
 7. The method ofscreening recombinant viruses containing desired genomic materials,wherein the virus is baculovirus, poliomavirus, papilomavirus, or HBV.8. A method of preparing recombinant viruses having circular genomicDNAs, which comprises the steps of: i) providing viral genomic DNAfragment containing at least two site-specific recombination sites andat least one restriction enzyme recognition site; ii) providing genecassette containing desired genomic materials flanked with at least twosite-specific sites; iii) reacting DNA fragment of i) and gene cassetteof ii) in vitro to produce reaction mixture including expressivecircular recombinant viral vectors; and iv) transferring the reactionmixture of the iii) to host cells in multi-well plate.
 9. The method ofpreparing recombinant viruses having circular genomic DNAs as claimed inclaim 8, wherein the viral genomic DNA fragment is non-infectious toanimal cells or cannot replicate and cannot form viral particles, whileincludes bacterial replication origin and antibiotics resistance DNAs.10. The method of preparing recombinant viruses having circular genomicDNAs as claimed in claim 8, wherein the transfer of the reaction mixtureto host cells is carried out in multi-well plate.
 11. The method ofpreparing recombinant viruses having circular genomic DNAs as claimed inclaim 8, wherein viral genomic DNAs of i) are digested with restrictionenzyme at one or more sites to generate linear formed viral genomicDNAs.
 12. The method of preparing recombinant viruses having circulargenomic DNAs as claimed in claim 11, wherein the restriction enzymerecognizes base sequences of CCTNAGG.
 13. The method of preparingrecombinant viruses having circular genomic DNAs as claimed in claim 8,wherein the virus is baculovirus, poliomavirus, papilomavirus or HBV.14. A recombinant virus prepared according to any one of methods ofclaims 8-13.